Novel methods for the assay of troponin i and t and complexes of troponin i and t and selection of antibodies for use in immunoassays

ABSTRACT

Assay systems and specialized antibodies for the detection and quantitation of troponin I and troponin T in body fluids as an indicator of myocardial infarction. Since troponin I and T exist in various conformations in the blood, the ratios of the monomeric troponin I an T and the binary and ternary complexes, as well as which form of troponin present in the blood, may be related to the metabolic state of the heart. Disclosed is a system to determine the presence of a troponin form or a group of troponin forms in a sample of whole blood, serum or plasma. 
     Disclosed is a stabilized composition of troponin; the stabilized composition can comprise a stabilized composition of troponin I, wherein the troponin I is oxidized, the troponin I can be unbound or the troponin I can be in a complex. 
     Disclosed is a method for improving the recovery of troponin I or T from a surface used in immunoassays. 
     Also disclosed are antibodies which recognize, unbound troponin forms, the forms of troponin in binary complexes, the ternary complex of troponin I, T and C, and the conformations of troponin I having intramolecularly oxidized and reduced cysteines.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/850,954, filed May 21, 2004, which is a Continuation of applicationSer. No. 09/687,051, filed Oct. 12, 2000, which is acontinuation-in-part of U.S. application Ser. No. 08/633,248, filed Apr.18, 1996, which is a Continuation in part of U.S. application Ser. No.08/423,582, filed Apr. 18, 1995, from each of which priority is claimed,and each of which is fully incorporated by reference herein.

TECHNICAL FIELD

This invention relates to the assay of troponin I and troponin T andcomplexes of these proteins, and more specifically to the changes inconformation of these proteins in blood, serum and plasma and to theselection of antibodies to the various forms of these proteins and theiruse in immunoassays. In another aspect of the invention, compositionsare taught for the stabilization and recovery of troponin I and T andtheir complexes in immunoassays.

BACKGROUND ART

Myocardial infarction is one of the leading causes of death in theUnited States. Approximately 5 million individuals experiencing chestpain are evaluated every year in hospitals throughout the United States,however, less than 30%, of these individuals are subsequently found tohave had a myocardial infarction. The accurate and rapid diagnosis ofmyocardial infarction is important both for the patient suffering amyocardial infarction and for the health care system which can minimizethe costs incurred by rapidly identifying individuals who do needtreatment.

The diagnosis of myocardial infarction is usually performed in theemergency department of a hospital. An individual haying the symptoms ofmyocardial infarction is treated in different ways depending on theobviousness of the condition. Generally, an electrocardiogram is givento assess the condition of the heart; however, approximately 50% ofpatients experiencing myocardial infarction have a non-diagnosticelectrocardiogram. The physician is then faced with a problem ofdiagnosing and treating the patient suspected of having a myocardialinfarction. Thus, diagnosis and treatment is difficult for patients witha suspected myocardial infarction who have non-diagnosticelectrocardiograms.

The World Health Organization (WHO) has instituted guidelines fordiagnosing myocardial infarction which state that an individual mustexhibit two-of-the-three following criteria: 1) have chest pain or ahistory of cardiac disease; 2) a diagnostic electrocardiogram; and, 3)elevated creatine kinase (CK) creatine kinase MB isoenzyme (CKMB). Thus,for the 50% of the individuals who are presented to hospitals for asuspected myocardial infarction and who have a non-diagnosticelectrocardiogram, the physician must rely on symptoms of chest pain andan elevated CK or CKMB to diagnose a myocardial infarction.

The assay of CK or CKMB is generally performed in hospital laboratoriesusing sophisticated instrumentation. The assays include enzyme assaysand immunoassays which detect the activity or mass of CK or CKMB presentin blood samples.

During a myocardial infarction, heart muscle cells die and release theircontents to the blood stream. The CKMB is released among such cellularcomponents. CKMB becomes elevated above an otherwise nominal value andcan be diagnostic fix myocardial infarction. The specificity of CKMBliar diagnosing myocardial infarction is not 100% because another sourceof CKMB in the body is skeletal muscle. Since the mass of skeletalmuscle in the body far exceeds the mass of cardiac muscle, through thenormal catabolic turnover of skeletal muscle cells, the bloodconcentration of CKMB in healthy individuals will vary. In general, theconcentration of CKMB which may be indicative of myocardial infarctionis above 5-7 ng/ml (Circulation 87, 1542-1550 (1993), Clin. Chem. 39,1725-1728 (1993)). The CKMB concentration of individuals who haveskeletal muscle injury or who have exercised has been reported to beelevated above 9 ng/ml (Clin. Chem. 38, 2396-2400 (1992)). Therefore,the problem of specificity when using CKMB as a marker for myocardialinfarction has prompted the search for other more specific markers whichare released only from damaged heart muscle.

Troponin I and troponin T have recently been shown to be more specificthan CKMB for diagnosing myocardial infarction (Circulation 83, 902-912(1991), Clin. Chem. 40, 1291-1295 (1994). Although troponin T has somedisadvantages as a marker because it is elevated in patientsexperiencing renal disease (Clin. Chem. 41, 312-317 (1995)), theinventive methods herein disclose the successful use of troponin T as adiagnostic marker. The use of troponin I as a diagnostic marker formyocardial infarction also appears to meet many of the clinicalrequirements (Clin. Chem. 40, 1291-1295 (1994), Clin. Chem. 41, 312-317(1995)).

The troponin complex in muscle is comprised of troponin I, C and T.These troponin components exist as various tissue specific isoforms.Troponin C exists as two isoforms, one from cardiac and slow-twitchmuscle and one from fast-twitch muscle. Troponin I and T are expressedas different isoforms in slow-twitch, fast-twitch and cardiac muscle(Biochem. J. 171, 251-259 (1978), J. Biol. Chem. 265, 21247-21253(1990), Hum. Genet. 88, 101-104 (1991), Circul. Res. 69, 1226-1233(1991)). The unique cardiac isoforms of troponin I and T allow them tobe distinguished immunologically from the other troponins of skeletalmuscle. Therefore, the release into the blood of troponin I and T fromdamaged heart muscle has been related to cases of unstable angina andmyocardial infarction. The prior art, however, has not addressed otherforms of troponin I and T in blood.

The troponin complex in muscle is tightly bound to the contractileapparatus. Approximately 6% of the troponin T in cardiac tissue existsas an unbound protein in the cytoplasm and it is believed that this poolof troponin T is released from damaged muscle (Am. J. Cardiol. 67,1360-1367 (1991)).

The conformations of troponin I, T and C change upon binding whenforming binary and ternary complexes (Biochemistry 33, 12800-128.06(1994). J. Biol. Chem. 254, 350-355 (1979), Ann. Rev. Biophys. Biophys.Chem. 16, 535-559 (1987)). Au understanding of the conformationalchanges of troponin I and troponin T and the heterogeneity of theproteins in the blood is critical for the development of accuratediagnostic procedures for measuring troponin I and troponin Tconcentrations. In addition, troponin I is reported to be unstable inblood (Direction Insert for Troponin I immunoassay, Sanofi/ERIADiagnostics Pasteur, Marries la Coquette, France), and the mechanismsresponsible for the instability have not been understood. This inventionaddresses these problems and provides for stable troponin I and Tcompositions which are useful in immunoassays.

The teachings of the instant invention provide methods for the selectionof antibodies and their use in immunoassays for troponin I and troponinT and complexes of these proteins. These proteins, along with troponinC, exist in both cardiac and skeletal muscle mainly as a ternarycomplex. In the muscle, the troponin complex is bound to tropomyosinwhich is, in turn, bound to the actin comprising the thin filaments. Thestate of troponin I and troponin T, whether free or bound as binary orternary complexes, which are released from the muscle, has not beenpreviously investigated.

DISCLOSURE OF THE INVENTION

Disclosed is an immunoassay system for determining the presence oramount of a troponin form or a group of troponin forms in a whole blood,plasma or serum sample suspected of containing troponin from damagedheart muscle. The system comprises: a) formation of an antibodyconjugate comprising an antibody coupled to a signal generating element,said antibody capable of specifically binding to cardiac specificregions of a form of troponin or a group of troponin forms; b) formationof a reaction mixture comprising said whole blood, plasma or serumsample incubated with said antibody conjugate; c) application of saidreaction mixture to a surface to which is bound at least one captureantibody, capable of specifically binding to cardiac specific regions ofa form of troponin or a group of troponin forms in said antibodyconjugate, said capture antibody binding said antibody conjugate,whereby the immobilized conjugate produces a detectable signal uponformation of sandwich complexes; and, d) relation of detectable signalto the presence or amount of said troponin form or said group oftroponin forms in said sample.

Also disclosed are antibodies that are sensitive and antibodies that areinsensitive to the form of troponin. An “antibody” refers to: amonoclonal antibody, a polyclonal antibody, a binding fragment of anantibody, a recombinant antibody, or a receptor protein thatspecifically binds to a target. As used herein, an insensitive antibodyis an antibody that yields an assay response that is less than withinabout a factor of 2 (i.e., 50% of the base value); and preferably yieldsan assay response that is within about 20% for each form of troponin,measured relative to assay response for the use of that antibody in anassay for a troponin form or group of troponin forms (the base value).Thus an insensitive antibody is one that will tend to hind more than oneform of troponin.

As used herein, it sensitive antibody in an immunoassay is one thatyields an assay response that is greater by at least about a factor of 2larger (i.e. 200% of the base value) and preferably a factor of 5 larger(i.e., 200% of the base value), for one or a group of forms of troponin(the base value), as compared to the assay response for other formsmeasured. Thus, a sensitive antibody is one that will tend to bend onlya single form of troponin.

As used heroin the nine troponin forms are: 1) the cardiac tertiarycomplex; 2) the cardiac troponin binary complex of I(oxidized)/T; 3) thecardiac troponin binary complex of I(reduced)/T; 4) the cardiac troponinbinary complex of I(oxidized)/C; 5) the cardiac troponin binary complexof I(reduced)/C; 6) the cardiac troponin binary complex T/C; 7) unboundcardiac troponin (oxidized); 8) unbound cardiac troponin I (reduced);and, 9) unbound cardiac troponin T.

Disclosed is a stabilized composition of troponin. The stabilizedcomposition can comprise a stabilized composition of troponin I, whereinthe troponin I is oxidized, the troponin I can be unbound or thetroponin I can be in a complex. The stabilized composition can comprisea stabilized composition of the ternary complex of troponin I, T and C.

Disclosed is a method for improving the recovery of troponin I or T froma surface used in immunoassays, said method comprising: contacting withsaid surface at least one strongly basic peptide protein, or polymerwith, a pI value greater than about 8; the method can further comprise astep of washing unbound peptide, protein or polymer from said membrane.Melittin can be the strongly basic peptide used, protamine can be thestrongly basic protein used.

DESCRIPTION OF FIGURES

FIG. 1 a illustrates the kinetics of air oxidation of troponin I asmeasured by immunoassay,

FIG. 1 b illustrates the kinetics of oxidation by peroxide of troponin Ias measured by immunoassay.

FIG. 2 illustrates the kinetics of reduction by dithiothreitol oftroponin I and reoxidation of reduced troponin I by peroxide as measuredby immunoassay.

FIG. 3 illustrates the effect of troponin C stat the immunoassay oftroponin I in the presence or absence of troponin T and bindinginhibitors.

FIG. 4 illustrates the kinetics of disruption of human cardiac troponinternary complex in the presence or absence of binding inhibitors asmeasured by an immunoassay for troponin I.

FIGS. 5 a-5 f illustrate the effect of binding inhibitors on troponin Iimmunoassays from patient samples with confirmed myocardial infarction.

MODES FOR CARRYING OUT THE INVENTION DEFINITIONS

As used herein, an “antibody” or “receptor protein” refers to amonoclonal antibody, a polyclonal antibody, a binding fragment of anantibody, a recombinant antibody, or a receptor protein thatspecifically binds to a target.

Specific binding of a substance signifies the quality of that substancethat the substance will tend not bind to something to which it does notspecifically bind; conversely, the substance will have greater affinityfor something it specifically binds that for a something it does notspecifically bind.

As used herein, an insensitive antibody in an immunoassay is an antibodythat for each form of troponin of interest yields an assay responsevalue that is the same within about a factor of 2, and preferably thesame within about 20%, as the assay response values for the other formsof interest. Thus, an insensitive antibody is one that will exhibit adetection of more than one form of troponin in an immunoassay.

As used herein, a sensitive antibody in an immunoassay is one that forone form or group of forms of troponin yields an assay response valuethat is at least about a factor of 2 larger, and preferably, about afactor of 5 larger, than the assay response values for other forms.Thus, a sensitive antibody is one that will exhibit a preferentialdetection of one form or group of forms of troponin in an immunoassay.

As used herein the nine troponin forms are: 1) the cardiac ternarycomplex; 2) the cardiac troponin binary complex of I(oxidized)/T; 3) thecardiac troponin binary complex of I(reduced)/T; 4) the cardiac troponinbinary complex of I(oxidized)/C; 5) the cardiac troponin binary complexof I(reduced)/C; 6) the cardiac troponin binary complex T/C; 7) unboundcardiac troponin I (oxidized); 8) unbound cardiac troponin I (reduced);and, 9) unbound cardiac troponin T.

As used herein, a “zone” is a concept that correlates with the abilityto identify distinct sensible signals. A zone, therefore, can correspondto a geographic region or correspond to the ability to separatelyidentify distinct sensible signals. The sensible signals can be distinctby, but not limited to, variations between the followingcharacteristics: wavelength of fluorescence or optical absorbance orreflectance; life time of or transition energy between, electronicstates; oxidation-reduction potentials; colorimetric characteristics;or, signal type (e.g., fluorescence vs. radioactivity vs. opticalabsorbance).

As used herein, unbound troponin is troponin that is not in a complex. Atroponin complex can be binary or ternary.

As used herein, a “label”, “signal generator” or “signal generatingelement” is an entity that can embody a number of different forms:Enzymes and their resultant effects on a substrate, colloidal metalparticles, latex and silica particles with dye incorporated, and dyeparticles are examples of signal generators. An enzyme can react on asubstrate to produce a product that is sensible, for example, bywavelength of fluorescence (e.g., ultraviolet, visible, infrared), orsensible by affect on pH.

Modes:

This invention is directed to the assay of troponin I and troponin T andcomplexes of these proteins in body fluids, particularly, in humanblood, serum and plasma. The presence of cardiac trope in I and T in theblood, above a nominal concentration, is diagnostic for damaged heartmuscle. The teachings of this invention show that troponin I and T existin various conformations in the blood which may be the same or differentthan their native conformations in muscle tissue. These variousconformations of the troponin molecules can react differently withantibodies.

The ratios of the monomeric troponin I and T and the binary and ternarycomplexes may be related to the metabolic state of the heart. Based onthe reactivities of antibodies to troponin I and T and to purifiedcomplexes of the troponins, the concentrations of troponin I and T andtheir complexes can now be elucidated in blood samples from patientssuffering from myocardial infarction.

The embodiments of this invention relate to the conformations oftroponin I and T and their complexes in blood, serum and plasma, and toantibodies which recognize those conformations. Specifically, antibodieswhich recognize troponin I and T in the following forms arepreferred: 1) The conformations of troponin I having intramolecularlyoxidized and reduced cysteines; 2) The binary complexes of troponin Iand T, of troponin I and C, of troponin T and C; and, 3) The ternarycomplex of troponin I, T and C. In addition, methods are described forthe improved recovery of troponin I and T in immunoassays. Thisinvention answers the heretofore unmet need for the assays of troponin Iand T in blood.

The focus on troponin I and T for use as markers for myocardialinfarction has been based in part on their molecular size: because theproteins are relatively small, it is believed that they leak out ofdamaged cells faster than the larger proteins.

Antibodies to Troponin Complexes and to Troponin I and T

The term “antibodies or receptor proteins” as used herein refer tomonoclonal and polyclonal antibodies, binding fragments of antibodies,and receptor proteins that specifically bend to a target. In a preferredembodiment, receptor proteins, for example, antibodies or bindingfragments, are directed to the epitopes of troponin I which areinsensitive to the oxidation state of the molecule. The terms sensitiveand insensitive herein refer generally to the ability of an antibody torecognize particular forms of free troponin or troponin complexes. Inparticular, a sensitive antibody which is useful in an immunoassaydistinguishes one form or forms of troponin from another form and aninsensitive antibody which is useful in an immunoassay does notdistinguish one form or forms of troponin from another. The sensitivityor insensitivity of an antibody is exhibited in an immunoassay. Todetermine whether an antibody is sensitive or insensitive, the antibodyis tested with each troponin form independently to yield the assayresponse of the antibody for each troponin form. In general, a preferredantibody that is insensitive to the troponin form will yield an assayresponse that is the same, within about a factor of two and preferablywithin 20%, for each form of troponin. A preferred antibody that issensitive to the troponin form will yield an assay response that is atleast a factor of two, and preferably a factor of five, larger for oneform or group of forms as compared with the other form(s). In addition,the terms “troponin I and T” can refer to the free, uncomplexedtroponins or to the troponins in the binary or ternary complexes. Humancardiac troponin I contains two cysteines, at positions and 97 (FEBSLetters, 270, 57-61 (1990)). In the current art, during the purificationof troponin I from tissues, the oxidation state of troponin I isdirected toward the reduced form using various reductants, includingmercaptoethanol, dithiothreitol and the like (Can. J. Biochem. 54,546-553 (1976), Methods. Enzymol. 85, 241-263 (1982)). Afterpurification, the current art also teaches to maintain troponin I in thereduced form to prevent intermolecular disulfide formation (J. Biol.Chem. 258, 2951-2954 (1983)).

In the development of immunoassays for a target protein, the purifiedtarget protein acts as a standard with which to judge the sensitivityand specificity of the immunoassay using the antibodies that have beenselected. As disclosed herein, the cysteines troponin I can rapidlyoxidize, intramolecularly, to alter the conformation of the protein. Thedegree of oxidation of troponin I has not previously been addressed withrespect to its effect on the immunoassay process. The teachingsdescribed herein show that an apparent instability in the troponin Imolecule is related to the dynamics of the intramolecular oxidation orreduction of the troponin I molecule. In addition, the selection ofantibodies is taught for the accurate quantitation of troponin I inblood.

Purified, reduced troponin I undergoes an intramolecular oxidation ofthe cysteines, the rate of which is not dependent on the troponin Iconcentration. Special care should be exercised when preparing theoxidized troponin I form, especially in the presence of various thiolreducing agents, because of the possibility of firming mixed disulfidesof the protein and the reducing thiol reagent. The mixed disulfide formof the protein may not behave as either the oxidized or reduced form ofthe molecule, especially if the antibodies used in the immunoassay bindto the region of the protein surrounding cysteines 80 and 97.

Using purified preparations of oxidized and reduced troponin I,differential effects in the immunoassays using various antibodies raisedto troponin I were observed. With some antibody pairs, the reducedtroponin I was hardly detectable in immunoassays, whereas with otherpairs, the oxidation state had no effect on the immunoassay process.These results showed that selection of antibodies to the troponin Imolecule, without prior knowledge of the oxidation state of the troponinI, can result in antibodies and an immunoassay process which giveserroneous results. This conclusion is exemplified by immunoassays oftroponin I from patients suffering myocardial infarction. The degree ofoxidation of troponin I in patient samples is variable and suggests apossible basis for apparent instabilities of troponin I assays of thecurrent art. The free troponin I does change its oxidation state fromthe reduced to the oxidized form over time (see Example 4).

In the case when an immunoassay comprising an insensitive antibody is tobe utilized to bind to the free and complexed troponin, a preferredantibody is one that is insensitive with respect to the oxidized,reduced and complex tones because the circulating troponin in the bloodcan change its oxidation state and the degree of binding to othertroponin components over time. For example, if reduced, troponin I isreleased from the heart, it can transform to the oxidized form duringcirculation in the blood and until the time troponin is measured. Inaddition, the free troponin I and T in the blood can bind to each otherand to troponin C to form binary and ternary complexes. The outcome ofthese transformations to the oxidized form of troponin I or to complexesof troponin is that when an antibody is chosen for an immunoassay thatis sensitive with respect to the oxidized and reduced forms andcomplexed forms, the assay will show an apparent increase or decrease inthe troponin concentration over time depending on which form (free,oxidized or reduced or complexed) the antibody better recognizes, ratherthan an actual change in troponin concentration. In another example, therelease of troponin complexes from damaged heart muscle can result inthe formation of free (uncomplexed) and binary forms of troponin I and Tduring circulation in the blood or until the troponin is assayed. Thevariability of assay results of troponin concentrations usingimmunoassays comprising a sensitive antibody or antibodies can mislead aphysician into believing that a patient's condition is improving ordeteriorating.

to the case when an immunoassay comprising more than one sensitive as isused to measure the free and complexed troponin, each sensitive antibodyis intended to be reacted with one form or group of forms of troponinsuch that the antibody exhibits a maximum assay response for theintended form(s) and a minimum assay response, for all other forms.Preferred antibodies tire ones for which the assay responses for theintended forms are about the same, preferably within 20%, for all thesensitive antibodies and said minimum assay responses are at least afactor of 2 and preferably is factor of 5 less than the maximum assayresponses. For example, if two different antibodies are used for signalantibodies in an immunoassay, and one antibody is insensitive withrespect to the oxidized and reduced free troponin I (or the freetroponin T) and exhibits a maximum assay response for said free forms oftroponin and a minimum assay response for the complexed troponin, thenthe other antibody should exhibit as maximum assay response for thecomplexed troponin and a minimum assay response for the free troponin Iand T forms. In this wad, an accurate measure of total troponin can bedetermined. One skilled in the art will also recognize that antibodieswith different affinities that is, exhibit different assay responses,for the troponin forms can also be utilized in immunoassays when eachtroponin form is measured alone or in discrete zones and that therelative bias of the immunoassays can be accounted for in thecalibration of the assay. Also, sensitive and insensitive antibodies canbe attached to solid phases to measure each troponin form in discretezones.

In another preferred embodiment, antibodies or binding fragments thatare directed to the epitopes of the troponin I or T are insensitive withrespect to free troponin I or T and troponin complexes. The teachingsherein show that antibodies that are sensitive with respect to treetroponin I or T and troponin complexes provide methods for theestimation of the extent of binding of troponin I or T in complexes.Using purified preparations of troponin I, T and C, and the purifiedtroponin complexes, the effects of troponin I or T binding in complexeson the recognition of troponin by antibody pairs is taught and isrelated to the dynamic state of troponin I or T in blood.

The degree of binding of troponin I and T to the components of thetroponin complex or to other proteins of the contractile apparatus,including tropomyosin and actin, in blood can also be problematic forimmunoassays depending on the degree and affinity of binding. In theirnative forms, the troponin complex exists in cardiac muscle and in slow-and fast-twitch skeletal muscle as a ternary complex of troponin I, Cand T. Troponin I and T from skeletal muscle have different amino acidsequences than troponin I and T from cardiac muscle, respectively;however, troponin C from slow-twitch muscle has the same amino acidsequence as the cardiac muscle protein (Nature 271, 31-35 (1978), Arch.Biochem. Biophys. 186, 411-415 (1978). FEBS Lett. 292, 5-8 (1991)). Thefast-twitch skeletal muscle troponin C, although not identical to thecardiac troponin C, can bind to cardiac troponin I (Biochemistry 33,8464-8471 (1994), Proc. Natl. Acad. Sci. USA 90, 9036-9040 (1993)).

The release of troponin components, that is, troponin C and T, orcomponents from the contractile apparatus, for example, tropomyosin andactin, from skeletal muscle, due to the normal turnover of skeletalmuscle cells; may result in a significant amount of troponin andcontractile apparatus components in the blood. Since skeletal musclemass is much greater than cardiac muscle mass, the troponin componentspresent in the blood of a normal individual may be derived largely fromskeletal muscle. The circulating troponin components which are mainlyderived from skeletal muscle would bind to cardiac troponin I and whichare, released into the blood during a myocardial infarction or eventswhich lead up to creating damaged heart muscle. As muscle damageprogresses in an individual the troponin components derived from hearttissue will presumably rise in the blood. Thus, the concentration oftroponin components (bound and free) in the blood from individualsexperiencing a myocardial infarction may be differentially derived fromboth cardiac and skeletal muscle.

The form of troponin released from the heart, whether free or as binaryor ternary complexes, into the blood may indicate a particular conditionor the heart. The assays taught herein provide for the analysis ofrelease patterns which may allow the physician to diagnose a specificheart failure, for example, unstable angina as compared to myocardialinfarction or to determine the time that an infarction occurred.

The clinical impact of an immunoassay measuring only the free troponin Ior T from a patient experiencing a myocardial infarction can be verysignificant. Since the binding of troponin I and T to troponincomponents in the blood will be variable, depending on the troponincomponent concentrations, and on the time that has elapsed since therelease of the troponin components from muscle an analysis of the boundand free form of the troponin I and T in the blood must be considered.For example, the binding affinity of troponin I to troponin C, in thepresence of calcium (which also is present in blood) is 1.27×10⁸ M⁻1(Biochemistry 33, 12729-12734 (1994)). This implies that if the troponinC concentration is 100 ng/ml and the total (bound and free) troponin Iconcentration is 8 ng/ml, then the free concentration of troponin I iscalculated to be 4.6 ng/ml. If the concentration of troponin I which isindicative of a myocardial infarction is 5 ng/ml or greater, then anassay which measures only the free form of troponin I, in this case, 4.6ng/ml, will indicate to the physician that a myocardial infarction hasnot taken place. Generally, in hospital emergency departments whichadmit patients believed to have had a myocardial infarction, a bloodsample from the individual will be obtained again in an hour or two ifthe first result is negative. In this example, the patient, having atotal troponin I concentration of 8 ng/ml in the first sample, (which isdefined as positive for a myocardial infarction), but only a measuredconcentration of 4.6 ng/ml, (which would be defined as a negativeresult), would not be treated and would continue to accrue damaged heartmuscle during the time before a second sample was analyzed.Interpretation of results of troponin T assays would also suffer fromtroponin T binding to components of the contractile apparatus in blood.Thus, immunoassays of the current art that measure the free troponin Iand T may not correctly diagnose a myocardial infarction when thetroponin I or T concentration, respectively, is near the decision point.In some cases, the rise or fall of the troponin I or T concentration ina patients blood over time as determined by analyzing blood samplesdrawn at several different times might be used to diagnose the dynamiccondition of the heart, for example, to determine whether the damagedheart is improving with therapy or continuing to deteriorate. In thesecases, the time-course of the free troponin I or T concentration couldbe different than that of the total troponin (bound and free)concentration. One skilled in the art will realize that increasingconcentrations of all the troponin components in the blood will resultin an increasing fraction of bound troponin I and T relative to freetroponin. The concentration of total troponin (bound and free) may risetaster than the concentration of free troponin I and T. An assay thatmeasures the total troponin concentration (bound and free troponin I andT) would be more accurate in assessing progression of heart damage ascompared with an assay that measures only tree troponin I or T.

In a particularly preferred embodiment, antibodies or binding fragmentsare directed to the cardiac troponin complex. Specifically, antibodiesare directed to cardiac specific epitopes troponin I and T of thetroponin complex or of the troponin I/T, I/C and T/C interfaces in thecomplex. The teachings herein show that antibodies that are raked totroponin I and T can bind poorly to troponin I or T of the ternarycomplex. Furthermore, the teachings herein show that the troponincomplex exists in the blood of patients who have experienced myocardialinfarction. Methods are also described which teach one skilled in theart to assess the amount of troponin complex in the blood relative tothe free troponin I and T or binary complexes of troponin I and T, usingantibodies which bind to the free troponin molecules.

The equilibrium among the binary and ternary complexes of troponin andfree troponin I and T will be altered during the immunoassay processbecause of the binding of antibodies to the troponin components andcomplexes. The change in the mole fractions of the various speciesduring the immunoassay may be significant or insignificant and will be afunction of the antibody concentrations, the affinity of the antibodiesfor the troponin components and complexes, the association constants forthe troponin components and complexes and the time that the antibodiesare allowed to bind to the troponin. These variables can change theperceived concentration of troponin I and T and lead to erroneousconclusions about the troponin concentration. For example, if twoimmunoassays utilize different antibody pairs for performing a sandwichimmunoassay and their antibody concentrations and affinities fortroponin I or T are different, and if a proportion of the troponin I orT occurs in the sample as binary and ternary complexes, one may expectthat each immunoassay will give a different result. In addition, ifblood samples contain varying concentrations troponin C, then theproportion of troponin I and T that is bound to troponin C as a binarycomplex will differentially perturb each immunoassay.

The teachings of the instant invention demonstrate that the troponinternary complex is more stable to dissociation than the binary complexesof troponin.

In another preferred embodiment, antibodies or binding fragments aredirected to epitopes which are not changed by proteolytic degradation ofthe N-terminal region of troponin I. The conformation of troponin I isalso reported to be affected by phosphorylation/dephosphorylation(Biophys. J. 63, 986-995 (1992), Biochem. 33, 12729-12734 (1994)). Inanother preferred embodiment, antibodies or binding fragments aredirected to epitopes of either troponin I or troponin I complexes, whichare or are not changed by the phosphorylation state of troponin I.Troponin I can be phosphorylated using methods described in J. Biol.Chem. 252, 851-857 (1977). The phosphorylated and dephosphorylatedpreparations of troponin I can be utilized as immunogens for generatingantibodies as well as antigens for the selection of antibodies to thephosphorylated and dephosphorylated troponin I. The troponin complex canbe dissociated into the component proteins using various treatments,including high concentrations of urea, low pH and metal chelating agentswhich bind divalent metal cations, particularly calcium and magnesium(Methods Enzymol. 85, 241-263 (1982)). These treatments are, in general,very harsh and require several hours. Thus, these conditions fordissociating the troponin complex are not practical for immunoassayswhich must be performed in a matter of minutes on samples fromindividuals who may be suffering a myocardial infarction.

The generation and selection of antibodies that are preferentiallyeither sensitive or insensitive to the binding, of troponin I or T inbinary complexes are accomplished by first preparing binary troponinI/T, T/C and I/C complexes from purified components (J. Biol. Chem. 254,350-355 (1979), J. Biol. Chem. 258, 2534-2542 (1983), J. Biol. Chem.258, 2951-2954 (1983), Can. J. Biochem. Cell Biol. 63, 212-218 (1985),Biochemistry 33, 12729-12734 (1994), Ann. Rev. Biophys. Biophys. Chem.16, 535-559 (1987)). The complexes may be stabilized, if necessary, bychemically cross-linking the proteins in the complex using methodsfamiliar to those skilled in the art. The generation and selection ofantibodies that are sensitive or insensitive to the binding of troponinI or T in the ternary complex can be accomplished several ways. Forexample, one way is to purify the ternary complex (Methods Enzymol, 85,241-263 (1983)) or to reconstitute the complex using the purifiedtroponin components. One skilled in the art will recognize that variousother contractile apparatus proteins which may be associated with thebinary or ternary complexes of troponins can also be constructed fromthe purified components and that the resultant complex can be utilizedto generate and select antibodies as taught by the instant invention.The complex can be stabilized with respect to dissociation by chemicallycrosslinking the components. The purified complexes are then injected,for example, into mice or rabbits, to generate polyclonal or monoclonalantibodies. Another way is to purify free (unbound) troponin I or T andthen inject the purified free troponin I or T, for example, into mice orrabbits, to generate polyclonal or monoclonal antibodies. One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies,Laboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments which mimicantibodies can also be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach (Borrebaeck, C.,ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)). In particular, the preparation, screening and selection ofrecombinant binding fragments is described in Examples 22 and 23.

The antibodies which are generated are selected by first screening foraffinity and specificity with the purified binary or ternary complexesand comparing the results to the affinity and specificity of theantibodies with the purified troponin I and T molecules for the desiredproperties which are defined by the immunoassay process.

The screening procedure can involve immobilization of the purifiedtroponin I or T or binary or ternary complexes or peptides to cardiacspecific sequences of the troponins in separate wells of microtiterplates. The solution containing a potential antibody or groups ofantibodies is then placed into the respective microtiter wells andincubated for about 30 min to 2 h. If an antibody to the protein ofinterest is present in the solution, it will bind to the immobilizedtroponin. In screening antibodies for binding to interfaces of binary orternary complexes of troponin, an antibody is first selected which bindsto the binary or ternary complex immobilized in the microliter well.That antibody is then further screened for its ability to bind to freetroponin components; that is, the potential interface antibody shouldnot bind to free troponin I, C or T which is immobilized in microtiterwells. In addition, the interface antibody should never be capable offorming a sandwich assay with binary or ternary complexes and anantibody which is known to bind to a specific troponin component in thecomplex in the presence of binding inhibitors which are known to disruptthe troponin complex. If this latter condition is met, then thepotential interface antibody should also not be capable of forming asandwich assay with the troponin complex and an antibody to a differenttroponin component than was used in the previous screen in the presenceof binding inhibitors. If this condition is also then an interfaceantibody has been selected for a binary complex. An extra immunoassaymust be performed for selecting an interface antibody to a ternarycomplex; that is, if the previous two conditions are met, then thepotential interface antibody should also not be capable of forming asandwich assay with the troponin complex and an antibody to a differenttroponin component than was used in the two previous screens. Themicrotiter wells are then washed and a labeled secondary antibody (forexample, an anti-mouse antibody conjugated to alkaline phosphatase ifthe raised antibodies are mouse antibodies) is added to the wells andincubated for about 30 min and then washed. Substrate is added to thewells and a color reaction will appear where antibody to the troponin ispresent. The antibodies which are of interest are then further analyzedfor affinity and specificity to the cardiac specific molecules and forcomplementarity in forming sandwich complexes with the antigens. Thoseskilled in the art will recognize that many approaches can be taken inproducing antibodies or binding fragments and screening and selectingfor affinity and specificity for the various troponin antigens, butthese approaches do not change the scope of the invention.

Assays for Troponin Complexes and Uncomplexed Troponin I and T

A particularly preferred embodiment of this invention is directed to theassay of troponin I and troponin T, particularly immunoassays, whereinthe antibodies selected for the assay bind to cardiac specific sequencesof the ternary complex, of the binary complexes and of the uncomplexed(free) troponin I or T in order to measure the complexed (bound) andfree fractions of troponin I and T, respectively. The cardiac specificsequences of troponin I and T are described in FEBS Lett. 270, 57-61(1990) and Genomics 21, 311-316 (1994). A synthetic peptide comprised of14 amino acids which mimics a cardiac specific sequence of troponin Iand methods used to prepare antibodies to the peptide are described inan International Patent Application number PC/US94/05468.

The immunoassay can be formulated with a cocktail of antibodies to bindall the troponin complexes and the free troponin I and T. Alternatively,the immunoassay can be formulated with specific antibodies thatrecognize epitopes of the troponin I and T in the complexes and also theunbound troponin I and T. In addition, the immunoassay can be formulatedwith antibodies that bind epitopes at interfaces of the componentproteins in the complexes and antibodies that bind the unbound troponinI and T.

A preferred immunoassay for troponin I or T involves conjugation of anantibody or a cocktail of antibodies to a label or a signal generator toform an antibody conjugate(s), which are capable of binding to cardiacspecific regions the troponin complexes of troponin I or T and tounbound troponin I or T. One skilled in the art will recognize that asignal generator has many forms. Enzymes, colloidal metal particles,latex and silica particles with dye incorporated, and dye particles areexamples of signal generators. Antibodies can be conjugated to thesignal generators in a variety of ways using heterobifunctional reagentsas taught in the Pierce Catalog and Handbook, Pierce Chemical Co.,Rockford, Ill. and in Uniform Latex Particles by Leigh B. Bangs, SeragenDiagnostics Inc., Indianapolis, Ind. hereby incorporated by reference.Another antibody or cocktail of antibodies is immobilized on a solidphase, for example, a membrane as taught in BioTechniques 4, 272-283(1986, and the membrane is placed in a device, for example, as describedin U.S. Pat. Nos. 4,727,019 and 5,458,852. The immobilized antibody iscomplementary to the antibody conjugate. The immobilized antibody andconjugate antibodies form sandwich complexes with the troponin I or Tcomplexes and also form sandwich complexes with troponin I or T,respectively. A plasma or serum sample suspected of containing troponincomplexes or components from damaged heart muscle is mixed with theantibody conjugate to form a reaction mixture that is allowed toincubate. The reaction mixture is then applied to the aforementioneddevice. The sample flows through the membrane and the troponin complexesand components, bound to the antibody conjugates, bind to theimmobilized antibodies, and excess, unbound, antibody conjugate iswashed away with a wash buffer. The signal is developed and read, eithervisually or instrumentally.

A particularly preferred immunoassay for troponin I involves conjugationof at least two antibodies to a label or a signal generator to form anantibody conjugate. One of the conjugate antibodies is capable ofbinding to the troponin T component of the troponin ternary complexesand the other antibody is capable of binding to the free and binarytroponin I molecules. Another antibody or cocktail of antibodies isimmobilized on a solid phase, for example, a membrane, and the membraneis placed in a device, as described previously. The immobilized antibodyis complementary with the antibody conjugate antibodies to form sandwichcomplexes with either troponin I bound to troponin complexes or to theuncomplexed troponin I. A plasma or serum sample suspected of containingtroponin complexes or components from damaged heart muscle is mixed withthe antibody conjugate to form a reaction mixture which is allowed toincubate. The reaction mixture is then applied to the aforementioneddevice. The sample flows through the membrane and the troponin complexesand components, bound to the antibody conjugates, bind to theimmobilized antibodies and excess, unbound antibody conjugate is washedaway with a wash buffer. The signal is developed and read, eithervisually or instrumentally. In this assay procedure, the antibodyconjugate binds to the troponin I in ternary complexes through thetroponin T specific antibody and all free and binary troponin Imolecules through the troponin I specific antibody. The capture antibodyor antibodies on the solid phase bind antibody conjugates that are boundto free troponin I, and to troponin ternary complexes that containtroponin I.

A particularly preferred immunoassay for troponin I (oxidized andeduced) involves conjugation of at least two antibodies to as label or asignal generator to form an antibody conjugate. One of the conjugateantibodies is capable of binding to the oxidized troponin I and theother antibody is capable of binding to the reduced troponin Imolecules. Another antibody or cocktail of antibodies is immobilized ona solid phase in up to 2 discrete zones, for example, a membrane, andthe membrane is placed in a device, as described previously. Theimmobilized antibody is complementary with the antibody conjugateantibodies to form sandwich complexes with either oxidized troponin I orreduced troponin I. A plasma or serum sample suspected of containingtroponin components from damaged heart muscle is mixed with the antibodyconjugate to form a reaction mixture which is allowed to incubate. Thereaction mixture is then applied to the aforementioned device. Thesample flows through, the membrane and the oxidized and reduced troponinI, bound to the antibody conjugates, bind to the immobilized antibodiesand excess, unbound antibody conjugate is washed away with a washbuffer. The signal is developed and read, either visually orinstrumentally. In this assay procedure, the antibody conjugates bind tothe oxidized troponin I through the oxidized troponin I specificantibody and to the reduced troponin I through the reduced troponin Ispecific antibody. The capture antibody or antibodies on the solid phaseonly bind antibody conjugates that are bound to oxidized and reducedtroponin I. This immunoassay may have application in the estimation oftime after an infarction has occurred.

Another particularly preferred immunoassay for troponin I involvesconjugation of an antibody or a cocktail of antibodies to a label or asignal generator to form an antibody conjugate. The antibody conjugatebinds to either troponin I bound to troponin complexes or to theuncomplexed troponin I. Immobilized on a solid phase, for example, amembrane, in 3 discrete zones, are antibodies or cocktails of antibodieswhich bind the ternary complex, the binary complexes troponin I and thefree troponin I, and the membrane is placed in a device, as describedpreviously. For example, a troponin T antibody which binds to thetroponin T of the ternary complex is immobilized in one discrete zone, atroponin I antibody which binds to the troponin I binary complexes(troponin I/C and I/T) is immobilized in another discrete zone and atroponin I antibody which binds to only the uncomplexed troponin I isimmobilized in yet another discrete zone. The immobilized antibodies arecomplementary with the antibody conjugate antibodies to form sandwichcomplexes with complexed or uncomplexed troponin I, as defined by eachdiscrete zone. Alternatively, immobilized on a solid phase, for example,a membrane, in 2 discrete zones, are antibodies or cocktails ofantibodies which hind the troponin I complexes (binary and ternary) andthe free troponin I. For example, a troponin T antibody which binds tothe troponin T of the ternary complex and as troponin I antibody whichbinds to the troponin I of the binary complexes are immobilized in onediscrete, zone and a troponin I antibody which binds to only theuncomplexed troponin I is immobilized in the second discrete zone. Theimmobilized antibodies are complementary with the antibody conjugateantibodies to form sandwich complexes with complexed or uncomplexedtroponin I, as defined by each discrete zone. A further embodiment ofthis invention utilizes antibodies on the solid phase for detection oftroponin I complexes which bind to the interfaces of the binding domainsof troponin I/T and I/C. A plasma or serum sample suspected ofcontaining troponin complexes or components from damaged heart muscle ismixed with the antibody conjugate to form a reaction mixture which isallowed to incubate. The reaction mixture is then applied to theaforementioned device. The sample flows through the membrane and thetroponin complexes and components, bound to the antibody conjugate(s),bind to the respective immobilized antibodies in the discrete zones andexcess, unbound antibody conjugate is washed away with a wash buffer.The signal is developed and read, either visually or instrumentally. Inthis assay procedure, the antibody conjugate binds to the troponin I andthe troponin I binary and ternary complexes through the troponin Ispecific antibody or antibodies. The capture antibody or antibodies indiscrete zones on the solid phase bind the antibody conjugates that arebound to the uncomplexed troponin I or troponin complexes containingtroponin I as defined by each discrete zone. This immunoassay allowsquantification of the fractions of troponin I, namely, the complexed andthe uncomplexed fractions. The inventive teachings described herein showthat uncomplexed and complexed troponin exists in plasma and serumsamples from patients with confirmed myocardial infarction. Thedetermination of the complexed and uncomplexed troponin I fractions mayyield important clinical data relating to the type and extent of muscledamage, for example, from unstable angina or myocardial infarction or tothe success of thrombolytic therapy.

Another particularly preferred immunoassay measures the cardiac troponinternary complex, the cardiac troponin binary complexes (troponin I/T,T/C and I/C and, the free cardiac troponin I and T. This method involvesconjugation of antibodies or a cocktail of antibodies to as label or asignal generator to form an antibody conjugate. The antibodyconjugates-bind to either troponin T and I bound to troponin complexesor to the uncomplexed troponin T and I. Immobilized on a solid phase,for example, a membrane in 1 discrete zone, are antibodies or cocktailsof antibodies which bind the ternary complex the binary complexes oftroponin I and T and the free troponin I and T and the membrane isplaced in a device, as described previously. For example, a troponin Iantibody which binds to the troponin I of the ternary complex, up to 3different antibodies, each recognizing the interfaces of the bindingdomains of troponin I/T, T/C and I/C, a troponin I antibody which bindsto the free troponin I and a troponin T antibody which binds to the freetroponin T are immobilized in a discrete zone. The immobilizedantibodies are complementary with the antibody conjugate antibodies toform sandwich complexes with complexed and uncomplexed troponin T and I.A plasma or serum sample suspected of containing troponin complexes orcomponents from damaged heart muscle is mixed with the antibodyconjugate to form a reaction mixture and it is allowed to incubate. Thereaction mixture is then applied to the aforementioned device. Thesample flows through the membrane and the troponin complexes andcomponents, bound to the antibody conjugate(s), bind to the respectiveimmobilized antibodies in the discrete zone and excess, unbound antibodyconjugate is washed away with a wash buffer. The signal is developed andread, either visually or instrumentally. In this assay procedure, theantibody conjugates bind to the free troponin I and T, to the troponin Iand T of the binary complexes and the troponin I or T of the ternarycomplex. The capture antibodies in the discrete zone on the solid phasehind the antibody conjugates which are specific to the free troponin Iand T and to the troponin complexes. This immunoassay allowsquantification of the ternary troponin complex, the troponin T/C, I/T,I/C and the free troponin I and T. One skilled in the art will recognizethat the antibodies specific to the various troponin forms can beconjugated to one or more signal generators to form antibody conjugatesand the antibodies previously described for the conjugates can beattached to the solid phase in a discrete zone. The determination of thetotal troponin concentration may yield a more sensitive immunoassay fordamage to the heart muscle that will, in turn, allow a more rapiddiagnosis of unstable angina or myocardial infarction and thereforefaster administration of thrombolytic therapy.

A particularly preferred immunoassay for troponin T involves conjugationof at least two antibodies to a label or a signal generator to form anantibody conjugate. One of the conjugate antibodies is capable ofbinding to the troponin I component of the troponin complexes and theother antibody is capable of binding to the free and binary troponin Tmolecules. Another antibody or cocktail of antibodies is immobilized ona solid phase, for example, a membrane, and the membrane is placed in asdevice, as described previously. The immobilized antibody iscomplementary with the antibody conjugate antibodies to form sandwichcomplexes with either troponin T bound to troponin complexes or to theuncomplexed troponin T. A plasma or serum sample suspected of containingtroponin complexes or components from damaged heart muscle is mixed withthe antibody conjugate to form a reaction mixture and it is allowed toincubate. The reaction mixture is, then applied to the aforementioneddevice. The sample flows through the membrane and the troponin complexesand components, bound to the antibody conjugates, bind to theimmobilized antibodies and excess, unbound antibody conjugate is washedaway with a wash buffer. The signal is developed and read, eithervisually or instrumentally. In this assay procedure, the antibodyconjugate hinds to the troponin complexes through the troponin Ispecific antibody and all free and binary troponin T molecules throughthe troponin T specific antibody. The capture antibody or antibodies onthe solid phase bind antibody conjugates that are bound to free troponinT, and to troponin complexes containing troponin T.

Another particularly preferred immunoassay for troponin T involvesconjugation of an antibody or a cocktail of antibodies to a label or asignal generator to form an antibody conjugate. The antibody conjugatebinds to either troponin T bound to troponin complexes or to theuncomplexed troponin T. Immobilized on a solid phase, for example, amembrane, in 3 discrete zones, are antibodies or cocktails of antibodieswhich bind the ternary complex, the binary complexes of troponin T andthe free troponin T, and the membrane is placed in a device, asdescribed previously. For example, a troponin I antibody which binds tothe troponin I of the ternary complex is immobilized in one discretezone, a troponin T antibody which binds to the troponin T binarycomplexes (troponin I/T and C/T) is immobilized in another discrete zoneand a troponin T antibody which binds to only the uncomplexed troponin Iis immobilized in yet another discrete zone. The immobilized antibodiesare complementary with the antibody conjugate antibodies to formsandwich complexes with complexed or uncomplexed troponin T, as definedby each discrete zone. Alternatively, immobilized on a solid phase, forexample, a membrane, in 2 discrete zones, are antibodies or cocktails ofantibodies which bind the troponin I complexes (binary and ternary) andthe free troponin T. For example, a troponin I antibody, which binds tothe troponin I of the ternary complex and a troponin T antibody whichbinds to the troponin T of the binary complexes are immobilized in onediscrete zone and a troponin T antibody which binds to only theuncomplexed troponin I is immobilized in the second discrete zone. Theimmobilized antibodies are complementary with the antibody conjugateantibodies to form sandwich complexes with complexed uncomplexedtroponin T, as defined by each discrete zone. A further embodiment ofthis invention utilizes antibodies on the solid phase for detection oftroponin T complexes which bind to the interfaces of the binding domainsof troponin C/T and I/T. A plasma or serum sample suspected ofcontaining troponin complexes or components from damaged heart muscle ismixed with the antibody conjugate to limn a reaction mixture which isallowed to incubate. The reaction mixture is then applied to theaforementioned device. The sample flows through the membrane and thetroponin complexes and components, bound to the antibody conjugates,bind to the respective immobilized antibodies in the discrete zones andexcess, unbound antibody conjugate is washed away with a wash buffer.The signal is developed and read, either visually or instrumentally. Inthis assay procedure, the antibody conjugate binds to the troponin T andthe troponin T binary and ternary complexes through the troponin Tspecific antibody or antibodies. The capture antibody or antibodies indiscrete zones on the solid phase bind the antibody conjugates that arebound to the uncomplexed troponin T or troponin complexes containingtroponin T as defined by each discrete zone. This immunoassay allowsquantification of the fractions of troponin T, namely, the complexed andthe uncomplexed fractions. The inventive teachings described herein showthat uncomplexed and complexed troponin exists in plasma and serumsamples from patients with confirmed myocardial infarction. Thedetermination of the complexed and uncomplexed troponin T fractions mayyield important clinical data relating to the type and extent of muscledamage, for example, from unstable angina or myocardial infarction or tothe success of thrombolytic therapy.

Another particularly preferred immunoassay independently measures thecardiac troponin ternary complex, the cardiac troponin binary complexes(troponin I/T, T/C and I/C) and the free cardiac troponin I and T. Thismethod-involves conjugation of antibodies or a cocktail of antibodies toa label or a signal generator to form an antibody conjugate. Theantibody conjugates bind to either troponin T and I bound to troponincomplexes or to the uncomplexed troponin T and I. Immobilized on a solidphase, for example, a membrane, in 6 discrete zones, are antibodies orcocktails of antibodies which bind the ternary complex, the binarycomplexes of troponin I and T and the free troponin I and T, and themembrane is placed in a device, as described previously. For example, atroponin I antibody which binds to the troponin I of the ternary complexis immobilized in one discrete zone, 3 different antibodies, eachrecognizing the interfaces of the binding domains of troponin I/T, T/Cand I/C, are immobilized in 3 discrete zones, a troponin I antibodywhich binds to the free troponin I is immobilized in another zone and atroponin T antibody which binds to the free troponin T is immobilized inanother zone. The immobilized antibodies are complementary with theantibody conjugate antibodies to form sandwich complexes with complexedand uncomplexed troponin T and I, as defined by each discrete zone. Aplasma or serum sample suspected of containing troponin complexes orcomponents from damaged heart muscle is mixed with the antibodyconjugate to form a reaction mixture and it is allowed to incubate. Thereaction mixture is then applied to the aforementioned device. Thesample flows through the membrane and the troponin complexes andcomponents, bound to the antibody conjugate(s), bind to the respectiveimmobilized antibodies in the discrete zones and excess, unboundantibody conjugate is washed away with awash buffer. The signal isdeveloped and read, either visually or instrumentally. In this assayprocedure, the antibody conjugates bind to the free troponin I and T, tothe troponin I and T of the binary complexes and the troponin I or T ofthe ternary complex. The capture antibodies in discrete zones on thesolid phase bind the antibody conjugates which are specific to the freetroponin I and T and to the troponin complexes. This immunoassay allowsquantification of the ternary troponin complex, the troponin T/C, I/T,I/C and the free troponin I and T. The inventive teachings describedherein show that uncomplexed and complexed troponin exists in plasma andserum samples from patients with confirmed myocardial infarction. Thedeterminations of the individual troponin ternary complex, the troponinI and T binary complexes and uncomplexed troponin I and T fractions mayyield important clinical data relating to the type and extent of muscledamage, for example, from unstable angina or myocardial infarction or tothe success of thrombolytic therapy.

Another particularly preferred immunoassay independently measures thecardiac troponin ternary complex, the cardiac troponin binary complexes(troponin I/T, T/C, I/C, oxidized I/T and oxidized I/C) and the freecardiac troponin I (oxidized and reduced) and T. This method involvesconjugation of antibodies or a cocktail of antibodies to a label or asignal generator to form an antibody conjugate. The antibody conjugatesbind to either troponin T and I bound to troponin complexes or to theuncomplexed troponin I and T. Immobilized on a solid phase, for example,a membrane, in up to 9 discrete zones, are antibodies or cocktails ofantibodies which bind the ternary complex, the binary complexes oftroponin I and T and the free troponin I and T, and the membrane isplaced in a device, as described previously. For example, a troponin Iantibody which binds to the troponin I of the ternary complex isimmobilized in one discrete zone, 5 different antibodies, eachrecognizing the interfaces of the binding domains of troponin I/T, T/C,I/C, oxidized I/T and oxidized I/C are immobilized in up to 5 discretezones, a troponin I antibody which hinds to the free troponin I(oxidized and reduced) is immobilized in up to 2 zones and a troponin Tantibody which binds to the free troponin T is immobilized in anotherzone. The immobilized antibodies are complementary with the antibodyconjugate antibodies to form sandwich complexes with complexed anduncomplexed troponin T and I, as defined by each discrete zone. A plasmaor serum sample suspected of containing troponin complexes or componentsfrom damaged heart muscle is mixed with the antibody conjugate to form areaction mixture and it is allowed to incubate. The reaction mixture isthen applied to the aforementioned device. The sample flows through themembrane and the troponin complexes and components, bound to theantibody conjugate(s), bind to the respective immobilized antibodies inthe discrete zones and excess, unbound antibody conjugate is washed awaywith a wash buffer. The signal is developed and read, either visually orinstrumentally. In this assay procedure, the antibody conjugates hind tothe free troponin I and T, to the troponin I and T of the binarycomplexes and the troponin I or T of the ternary complex. The captureantibodies in discrete zones on the solid phase bind the antibodyconjugates which are specific to the free troponin I and T and to thetroponin complexes. This immunoassay allows quantification of theternary troponin complex, the troponin T/C, I/T, I/C, oxidized I/T andoxidized I/C and the free troponin I (oxidized and reduced) and T. Theinventive teachings described herein show that uncomplexed and complexedtroponin exists in plasma and serum samples from patients with confirmedmyocardial infarction. The determinations of the individual troponinternary complex, the troponin I and T binary complexes and uncomplexedtroponin I and T fractions may yield important clinical data relating tothe type and extent of muscle damage, for example, from unstable anginaor myocardial infarction or to the success of thrombolytic therapy.

A particularly preferred immunoassay for troponin measures theconcentration of two or more forms of troponin, for example of free andcomplexed troponin I and or T, utilizing antibodies that have varyingdegrees of recognition for the different forms of troponin. An antibodyor cocktail of antibodies are conjugated to a label or signal generatorto form an antibody conjugate. The antibody conjugate has the ability tobind to each form of troponin that is to be quantified. A preferredantibody for the conjugate would be an “insensitive” antibody as definedabove. Immobilized on a solid phase, for example, a membrane, in adevice, in discrete zones are antibodies or cocktails of antibodies thatare complementary to the antibody conjugate antibody(s). The essentialcharacteristics of the immobilized antibodies are defined in terms oftheir responses in the assay, and are, therefore, discussed below afterthe assay is described. The essential features of the assay can bediscussed for the case in which two terms of troponin are to bequantified. In the case when two forms, form 1 and 2, of troponin are tobe quantified, two discrete zones are utilized. The system is calibratedusing two sets of calibrators in a suitable matrix such as blood, plasmaor serum. Preferably one set of calibrators contains form 1 of troponinat various concentrations and the other contains form 2 of troponin.Independently, each of the calibrators is mixed with the antibodyconjugate to form a reaction mixture which is allowed to incubate. Thereaction mixture is then applied to the aforementioned device. Thesample flows through the membrane and the troponin, bound to theantibody conjugate(s), binds to the immobilized antibodies in thediscrete zones and excess, unbound antibody conjugate is washed awaywith a wash buffer. The signal on both zones 1 and 2 is developed andmeasured for each calibrator. The simplest and preferred system would beone in which the assay signal is linear, or can be approximated to belinear, with respect to the troponin concentration. In this case, thecalibration procedure would yield four independent assay slopes definedas follows:

m₁₁=assay slope determined on zone 1 using form 1 of troponin as thecalibrator  (Eqn. 1a)

m₁₂=assay slope determined on zone 1 using form 2 of troponin as thecalibrator  (Eqn. 1b)

m₂₁=assay slope determined on zone 2 using form 1 of troponin as thecalibrator  (Eqn. 1c)

m₂₂=assay slope determined on zone 2 using form 2 of troponin as thecalibrator  (Eqn. 1d)

and two independent assay constants defined as follows:

c₁=assay signal on zone 1 corresponding to zero troponinconcentration.  (Eqn. 2a)

c₂=assay signal on zone 2 corresponding to zero troponinconcentration.  (Eqn. 2b)

One skilled in the art will recognize that the slopes and constantsshown in Eqns. 1 and 2 could also be determined in a calibration inwhich the calibrator solutions are comprised of both forms of troponinat various different ratios of concentrations. A blood, plasma or serumsample suspected of containing forms 1 and 2 of troponin is then assayedas described, above for the calibrators. The assay yields two signalvalues: S₁ is the signal measured in zone 1 and S₂ is the signalmeasured in zone 2. The two signals are described by two independentlinear equations:

S ₁ =m ₁₁[form 1]+m ₁₂[form 2]+c ₁  (Eqn. 3a)

S ₂ =m ₂₁[form 1]+m ₂₂[form 2]+c ₂  (Eqn. 3b)

where [form 1] and [form 2] are the concentrations of form 1 and 2 oftroponin, respectively, in the sample. Equations 3 can be solved usingstandard techniques of linear algebra to determine values for [form 1]and [form 2] in the sample if:

m ₁₁ m ₂₂ −m ₁₂ m ₂₁≠0  (Eqn. 4)

(see for example, Mathematical Methods for Physicists, Acad. Press, NY,N.Y.). Equations 1 and 4 define, therefore, the assay responses that arerequired for the antibodies in zone 1 and zone 2 to determine theconcentrations of form 1 and form 2 of the troponin from the measuredsignals S₁ and S₂. In general, the accuracy of the determination of theconcentrations of form 1 and form 2 will increase with an increase inthe difference shown in Eqn. 4. The value of the difference required toobtain a satisfactory result will depend on the precision of thecalibration and the assay, and the accuracy required for the troponinconcentration. For most assay systems, a preferred assay is one in whichthe antibody or cocktail of antibodies in at least one of the zones issensitive to whether the troponin is form 1 or form 2, where “sensitive”is defined in a previous section. For example, either the ratio m₁₁/m₁₂or the ratio m₂₂/m₂₁ or both should be larger than about 2. Thisprocedure for determining the concentrations of two forms of troponincan be extended to more than two forms. If N forms are to be measured, Ndiscrete zones must be used. A calibration must be performed utilizing aminimum of N+1 calibrator solutions comprised of the N forms oftroponin. A sample suspected of containing the N forms of troponin isassayed. The assay signal from each of the N zones is measured, thesignal S_(i) from the i'th zone can be expressed as follows:

$\begin{matrix}{S_{i} = {{\sum\limits_{j = 1}^{j = N}\; {m_{ij}\lbrack{formj}\rbrack}} + c_{i}}} & \left( {{Eqn}.\mspace{14mu} 5} \right)\end{matrix}$

where m_(ij) is the assay slope determined on the i'th zone using form jof the troponin as the calibrator and [form j] is the concentration ofform j of the troponin the sample. Eqn 5 defines a set of N linearequations that can be solved to determine the concentrations of all Nthrills of troponin using standard techniques if the determinant of thematrix comprised of the m_(ij)'s is not equal to zero. For most assaysystems, a preferred assay is one in which at least N−1 of the zonescontain an antibody or cocktail of antibodies that is sensitive to whichform the troponin is in, i.e., the ratio m_(i)/m_(ij) where I≠j, is,greater than about 2. Finally, these concepts can be extended to thecase in which the assay response is not linear with the troponinconcentration. In this case, the signal measured on the i'th of N zoneswill be given by the relation:

$\begin{matrix}{S_{i} = {\sum\limits_{j = 1}^{j = N}\; F_{ij}}} & \left( {{Eqn}.\mspace{14mu} 6} \right)\end{matrix}$

where F_(ij) is a function of [form j] that describes the dose-response(Signal as a function of [form j]) of form j on zone 1 and that isdetermined in a calibration procedure similar to those described above.Equation 6 describes a system of N non-linear equations which may besolved to determine the concentrations of the N forms of troponin using,for example, approximation (computer) methods familiar to those skilledin the art.

in another embodiment of this invention, inhibitors which affect theaffinity constants of the association of troponin I complexes or oftroponin T complexes are added to the sample prior to or with formationof the reaction mixture so that the free troponin I or troponin T ismeasured, respectively. The binding inhibitors may disrupt the troponincomplexes or they may open up or partially unravel the complex, suchthat some or all interactions between the troponin components are brokenso that epitopes may be more easily assessable to the antibodies forbinding. Inhibitors can be selected front the group of compounds whichcomprise, but is not limited to, metal chelating agents and peptideswhich compete with troponin I or troponin T for binding to proteins ofthe contractile apparatus. Metal chelating agents, particularly thosewhich bind to calcium and magnesium, lower the affinity constant, forexample, of troponin I and troponin C binding by about a factor of 10 ascompared to the affinity in the presence of calcium (Biochemistry 33,12729-12734 (1994)). Peptides which affect troponin I and troponin Tbinding to proteins of the contractile apparatus include mastoparan,melittin and peptide sequences which mimic the troponin I and Tsequences at their binding domains with the proteins of the contractileapparatus ((Biochemistry 31, 11326-11334 (1992), J. Biol. Chem. 267,15715-15723 (1992), Biochemistry 13, 8233-8239 (1994)). Other peptideswhich are useful as inhibitors are those which mimic the binding domainsof the troponin components. The binding domains are described, forexample, Ann. Rev. Biophys. Biophys. Chem, 16, 535-559 (1987), and withthe binding domain information, one skilled in the art synthesizes thepeptide which mimics the peptide of the protein at the binding domain.

In another embodiment of this invention, troponin C is added to thesample prior to or with formation of the reaction mixture of theimmunoassay so that all or nearly all of the troponin I or T in thesample will be bound by troponin C during the course of the assay. Thetroponin C concentration in the sample should be about 0.5 μg/ml to 100μg/ml and preferably about 1 to 10 μg/ml.

In another embodiment of this invention, troponin C and T are added tosamples prior to or with formation of the reaction mixture ofimmunoassays for troponin I in order to bind all or nearly all of thetroponin I in the form of the ternary troponin complex. The troponin Cand T concentrations in the sample should be about 0.5 to 100 μg/ml andpreferably about 1 to 10 μg/ml.

In yet another embodiment of this invention, troponin C and I are addedto samples prior to or with formation of the reaction mixture ofimmunoassays for troponin T in order bind all or nearly all of thetroponin T in the form of the ternary troponin complex. The troponin Cand T concentrations in the sample should be about 0.5 to 100 μg/ml andpreferably about 1 to 10 μg/ml.

These embodiments wherein troponin components are added to the sampleprior to or with formation of the reaction mixture have severaladvantages.

Firstly, troponin I adsorbs tenaciously to glass surfaces and variousmembranes which can result in a lower measured troponin I concentration.Troponin T also adsorbs to surfaces. However, when bound to troponin Cor in the ternary complex, the adsorptive characteristics of troponin Iand T may be dramatically reduced. Thus, the recovery of the troponinI/C or T/C complex or the ternary complex can be better than troponin Ior T. In this embodiment, antibodies that recognize the troponin I or Tcomplexes are used in the immunoassays.

Secondly, if antibodies which bind only to the complexed troponin I or Tare required, then the antibody selection process is less stringentbecause the antibodies are not required to have a similar affinity tothe free troponin I or T.

One skilled in the art will appreciate the inventive teachings describedherein and will recognize with these teachings that addition of reagentsto a device or to each other, as recited in the embodiments, has manyforms, and these forms are within the scope of this invention.

Stabilization of Troponin I or T for Calibrator Reagents

Troponin I and T are known to be unstable in aqueous formulations, aswell as in patient samples. The (apparent) instabilities of theproteins, as taught herein, are related to the oxidation state of thetroponin I, the propensity of troponin I and T to form complexes withother troponin proteins and the adsorptive characteristics of troponin Iand T onto surfaces.

Stabilization of troponin I is performed by the intramolecular oxidationof the cysteines and the protein is stored without, thiol reducingagents, such as mercaptoethanol, dithiothreitol and the like.

The storage of troponin I in solutions containing high concentrations ofthiol reductants will maintain the cysteines, overall, in the reducedform. However, intramolecular oxidation and reduction of a protein is adynamic process whereby the protein will exist for some time in theoxidized form even in the presence of the reductants. In the case ofreductants, such as mercaptoethanol or N-acetylcysteine, that is,reductant molecules with only a single thiol group, mixed disulfides ofthe reductant and the protein thiol will form. The half-life of thismixed disulfide will be a function of the reductant concentration andthe rate of intramolecular oxidation; that is, the mixed disulfide canbe reduced by both the thiol reductant reagent and the other proteincysteine, assuming that both cysteines are not in the mixed disulfideform. In the case of reducing the intramolecularly oxidized troponin I,the reductant with a single thiol group will reduce the intramolecularcystine to yield a cysteine and a mixed disulfide of the protein. Themixed disulfide of the protein will be reduced by either the cysteine ofthe protein or the thiol reductant. This process continues andeventually the reductant concentration is depleted to a level where itcan no longer-maintain the protein in the reduced state. As thereductant concentration approaches the concentration of thiol in thetroponin I, the protein cysteine and the thiol reductant reagent canform mixed disulfides, which will not be reduced by the thiol reductant.Alternatively, the protein will oxidize, intramolecularly, and the thiolreductant is not in sufficient concentration to reduce the cystine. Theend result, upon depletion of the thiol reductant, will be a mixture oftroponin I which is in the intramolecularly oxidized form and proteinwhich is in the mixed disulfide form. Each of these forms of troponin Ihas a different conformation.

In the case of utilizing thiol reductant reagents which possess twothiol groups, for example dithiothreitol or dithioerythritol, the endresult, upon depletion of the thiol reductant, will be only the oxidizedform of the troponin I.

Therefore, antibodies which are sensitive to the oxidation state of thetroponin I will differentially recognize the various forms of thetroponin I in the immunoassay. The immunoassay will then measure aninaccurate concentration of troponin I.

A preferred composition of stabilized troponin I comprises an aqueoussolution of the intramolecularly oxidized troponin I. A particularlypreferred composition of stabilized troponin I and T comprises abuffered solution of the ternary complex of troponin I, T and C in thepresence or absence of calcium and magnesium salts. A preferred range ofpH of the solution is between 6 and 9 and a range of calcium andmagnesium salts concentrations, for example calcium and magnesiumchloride, of between 0.01 mM and 10 mM. A particularly preferredbuffered solution consists of up to about 100% human serum or plasma.The ternary complex can be formed from the component troponin I, T andC, or alternatively, it can be isolated and purified from cardiac orskeletal muscle (Methods Enzymol. 85, 241-263 (1982)).

Methods for Improving the Recovery of Troponin I in Membranes

The adsorption of troponin I and T to surfaces and to various proteinsis known to occur and this phenomenon can lower the measured troponinconcentration. In particular, when immunoassays are performed in devicesor instruments which have a large surface area, for example, whenmembranes or latex particles are incorporated into the assay process,the surface area which is exposed to the sample can lower the recoveryof troponin. Membranes made up of nylon or compositions of glass fibershaving sires of between 2 mm×2 mm×1 mm and 40 mm×40 mm×5 mm caninfluence the recovery of troponin I and T when coupled with the assayprocess.

The troponin I and T molecules have a high degree of basic amino acids.At physiological pH, the basic amino acids are largely positivelycharged and these charged groups contribute to the adsorptive behaviorof the proteins.

In a preferred embodiment of this invention, various components areadded to membranes or latex particles to improve the recovery oftroponin I and T in the immunoassay process. Specifically, peptidesproteins which are also strongly basic are added to membranes or latexparticles or surfaces of devices involved in the assay process prior toor with addition of the sample or reaction mixture. Preferred compoundsfor this use include peptides, proteins and polymers with pI valuesgreater than about 8. Included in this group are salmine, lysozyme,cytochromes, protamine, polylysine, polyvinyl amine, melittin andmastoparan. Concentrations of blocking reagents which are added tosurfaces or membranes range from about 0.01 mg/ml to 100 mg/ml andtypically about 0.1 mg/ml to 10 mg/ml.

EXPERIMENTAL SECTION Example 1 Preparation of Reagents for TroponinELISA Immunoassays Preparation of Anti-Troponin Antibody AlkalinePhosphatase Conjugates

Alkaline phosphatase (Calzyme, San Luis Obispo, Calif.) in 50 mMpotassium phosphate, 10 mM potassium borate, 150 mM sodium chloride, pH7.0, at 10 mg/ml was derivatized with SMCC (succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate, Pierce Chemical Co.,Rockford, Ill.) at a 15/1 molar ratio of SMCC/enzyme. The derivatizationwas performed at room temperature for 90 min and subsequentlychromatographed on a GH-25 column (Amicon Corp., Beverly, Mass.)equilibrated in 50 mM potassium phosphate, 10 mM potassium borate, 150mM sodium chloride, pH 7.0.

The anti-troponin antibodies in 50 mM potassium phosphate, 10 mMpotassium borate, 150 mM sodium chloride, pH 7.0, at 10 mg/ml werederivatized with SPDP (N-succinimidyl-3-[2-pyridyldithio]propionate,Pierce Chemical Co.) at a 10/1 molar ratio of SPDP antibody. Theantibody was diluted to 2 mg/ml and Dithiothreitol and taurine wereadded to the solution at final concentrations of 1 mM and 20 mM,respectively, and the solution was subsequently incubated at roomtemperature for 30 min. The antibody-SPDP was chromatographed on a GH-25column (Amicon Corp.) equilibrated in 50 mM potassium phosphate, 10 mMpotassium borate, 150 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid, pH 7.0.

The SMCC-alkaline phosphatase (in 50 mM potassium phosphate, 10 mMpotassium borate, 150 mM sodium chloride, 5 mM magnesium chloride, pH7.0) and the thiol-antibody in 50 mm potassium phosphate, 10 mMpotassium borate, 150 mM sodium chloride, 0.1 mM ethylenediaminetetraacetic acid pH 7.0), both diluted to 1 mg/ml, were rapidly added toeach other with mixing in equimolar amounts. The solution was incubatedat room temperature for 3 hours, after which N-ethyl maleimide was addedto a final concentration of 2 mM.

Preparation of Biotinylated Troponin Antibodies

Biotin-XX, succinimidyl ester(6-((6((biotinoyl)amino)hexanoyl)amin-o)hexanoic acid, succinimidylester, Molecular Probes, Eugene. Oreg.) at 40 mM in dimethylformamidewas added slowly with mixing to an antibody solution at 2 mg/ml in 50 mMpotassium borate, 150 mM sodium chloride, pH 8.2, (BBS) to achieve afinal molar ratio of 20/1 biotin-XX/antibody. The solution was incubatedat room temperature liar 2 h, after which the solution was dialyzed at4° C. for at least 12 h.

Preparation of Avidin-HS Magnetic Latex

One ml of Estapor Paramagnetic latex particles (0.94μ, BangsLaboratories, Carmel, Ind., at 10% solids, washed 4 times with deionizedwater) in water was added to 9 ml of 0.55 mg/ml avidin-HS (ScrippsLaboratories, San Diego, Calif.) in 50 mM Tris hydrochloride, 15.0 mMsodium chloride, pH 7.5. The latex solution was incubated at 45 C for 2h. The latex was washed 3 times, each with 10 ml BBS, and resuspended in10 ml BBS.

Example 2 Immunoassay of Human Cardiac Troponin I and Troponin T

Two immunoassay protocols are described. They were used to detectTroponin I and Troponin T, present in human serum, plasma or insolutions containing purified proteins.

Protocol A

The sample containing troponin I or troponin T was diluted to 1-10 ng/mltroponin I or troponin T an assay buffer (hereafter called assay buffer)containing 10 mM 3-(N-morpholino) propane sulfonic acid, 650 mM sodiumchloride, 1 mM magnesium chloride, 0.1 mM zinc chloride, 1 mg/mlpolyvinyl alcohol (1000 m.w.), 10 mg/ml bovine serum albumin, 1 mg/mlsodium azide, pH 7.0. To 25 μl of diluted sample in a microtiter platewell was added 50 μl of assay buffer containing 2.5 μg/ml anti-troponinI or troponin T antibody conjugates (Example 1) and 2.5 biotinylatedanti-troponin I or troponin T polyclonal antibody (Example 1) to form areaction mixture. After a 30 minute incubation of the reaction mixtureat room temperature, 25 μl of avidin-HS coated magnetic latex (Example1; 0.5% latex in assay buffer) was added to the microtiter plate well,followed has a 5 minute incubation at room temperature. The magneticlatex was pelleted using a microtiter plate magnet (PerceptiveDiagnostics, Cambridge, Mass.) and washed twice in BBS-Tween (20 mMborate, 150 mM sodium chloride, 0.1 mg/ml sodium azide, 0.02%Polyoxyethylene-20 Sorbitan Monolaurate (Tween-20), pH 8.2) and once inTBS (40 mM Tris, 150 mM sodium chloride, pH 7.5) The pellet wasresuspended in ELISA amplification reagents (Gibco BRL, Gaithersburg,Md.) according to the manufacturer's instructions. After theamplification was complete, the magnetic latex was pelleted and 80 μl ofthe colored supernatant was transferred to a fresh microtiter plate. Theabsorbance at 490 nm (OD₄₉₀) was measured using a microtiter platereader (Molecular Devices, Palo Alto, Calif.).

Protocol B

The sample containing troponin I or troponin T was diluted into assaybuffer as described in protocol A. To 80 μl of diluted sample in amicroliter plate well was added 40 μl of assay buffer also containing 30μg/ml anti-troponin I or troponin T monoclonal antibody and 7.5 μg/mlbiotinylated anti-troponin I or troponin T polyclonal antibody(Example 1) that was complimentary to the monoclonal antibody to formthe reaction mixture. Aliquots (25 μl) were removed at various times (2minutes to 24 hours) and were added to microtiter plate wells containing25 μl of avidin-HS coated magnetic latex (0.5% latex solids in assaybuffer), followed by a 5 minute incubation. The magnetic latex waspelleted and washed once in BBS-Tween and once in assay buffer. Thepellet was resuspended in 25 μl of assay buffer also containing 5 μg/mlof goat anti mouse kappa antibody conjugated to alkaline phosphatase(Southern BioTechnology Associates, Inc., Birmingham, Ala.) followed bya 15 minute incubation. The magnetic latex was pelleted and theremainder of the assay was performed as indicated in Protocol A.

Example 3 Selection of Anti-Troponin I Antibodies that Bind theOxidized, the Reduced or Both the Oxidized and Reduced Forms of HumanCardiac Troponin I

Anti-troponin I antibody conjugates (Example 1) and complimentarybiotinylated troponin I polyclonal antibodies (Example 1) were testedfor recognition of intramolecularly oxidized or reduced troponin I. Theanti troponin I monoclonal antibodies tested were: clone 2D5 and clone1A12 (BiosPacific, Emeryville, Calif.), clone 110 and 111 (ResearchDiagnostics, Inc., Flanders, N.J.) and clone TRI-7 F81 (DAKOCorporation, Carpinteria, Calif.). The biotinylated anti-troponin Iantibodies tested were affinity-purified goat polyclonals, specified aspeptide 1, peptide 2, peptide 3 or peptide 4 specific (BiosPacific,Emeryville, Calif.). Human cardiac troponin I, (P. Cummins. Universityof Birmingham, Birmingham, UK) was air oxidized at 1.0 μg/ml asdescribed in example 4 to form the intramolecular disulfide. Theoxidized troponin I was diluted to 1-10 ng/ml in assay buffer eitherwithout (oxidized sample) or with (reduced sample) dithiothreitol (DTT)at a final concentration of 3 mM, followed by a 3 hour incubation atroom temperature to allow reduction of the disulfide by DTT. Theoxidized and reduced samples were assayed without further dilution usingProtocol A of Example 2. The results are shown in Table 1 and areexpressed in terms of a ratio of the assay slope for oxidized troponin I(TnI) divided by the assay slope for reduced troponin I. The assay slopeincreases with increasing recognition of troponin I by the antibodypair.

The data show that antibodies can be selected that either preferentiallybind oxidized or reduced troponin I or bind oxidized and reducedtroponin I approximately the same. Selection of the antibodies withoutregard to the oxidation-reduction state of troponin I can lead to asubstantial error in the quantification of the troponin I concentration.

TABLE 1 Anti troponin I Anti troponin I Ratio of assay slopes monoclonalantibody polyclonal antibody (oxized TnI/reduced TnI) Clone 2D5 peptide1 8.3 specific Clone 2D5 peptide 3 10 specific Clone 1A12 peptide 1 0.6specific Clone 1A12 peptide 3 1.3 specific Clone 1A12 peptide 4 1.2specific Clone TRI-7 F81 peptide 1 0.5 specific Clone TRI-7 F81 peptide2 0.5 specific Clone TRI-7 F81 peptide 3 0.5 specific Clone TRI-7 F81peptide 4 0.5 specific Clone 110 peptide 4 0.8 specific Clone 111peptide 3 1.0 specific Clone 111 peptide 4 0.4 specific

Example 4 Oxidation-Reduction of Purified Human Cardiac Troponin I

The kinetics of intramolecular oxidation and reduction of purifiedtroponin I (P. Cummins, University of Birmingham, UK) was measured withan immunoassay (Protocol A, Example 2) using a clone 2D5 antibodyconjugate (Example 1) and biotinylated goat anti troponin I peptide 0.1polyclonal antibody (Example 1). This antibody pair binds strongly tooxidized troponin I and weakly to reduced troponin I as described inExample 3. The results of the assay are expressed in terms of an assayslope [OD₄₉₀ per ng/ml total (oxidized+reduced) troponin I] in thelinear range of the assay. The assay slope increases with the fractionof oxidized troponin I.

Air Oxidation of Reduced Troponin I

The rate of air oxidation of troponin I at two troponin I concentrationswas measured. Reduced troponin I at 0.27 mg/ml in a buffer containing 20mM Tris-HCl, 0.5 M sodium chloride, 60 mM 2-mercaptoethanol, pH 7.5, wasdiluted to either 1300 ng/ml or 10 ng/ml in assay buffer containingeither no or 25 mM 2-mercaptoethanol. The solutions were incubated atroom temperature. Aliquots were taken after various incubation times, asindicated in FIG. 1 a, diluted to 4 and 8 ng/ml troponin I in assaybuffer, and assayed immediately. The results are shown in FIG. 1 a,wherein the error bars represent 1 standard deviation (SD).

Peroxide Oxidation of Reduced Troponin I

Peroxide (Fisher, unstabilized) was added (final concentration ofperoxide 2 mM or 20 mM) to an aliquot of the 1300 ng/ml solution ofreduced troponin I (see this example, air oxidation) immediately afterthe troponin I was diluted from the 0.27 mg/ml stock solution. Thesolutions were incubated at room temperature. Aliquots were taken aftervarious incubation times, as indicated in FIG. 1 b, treated withcatalase (Calbiochem, La Jolla, Calif.; 0.01 mg/ml final concentrationfor 5 minutes) to remove the peroxide, diluted to 4 and 8 ng/ml troponinI, and assayed immediately. The results are shown in FIG. 1 b, whereinthe error bars represent 1 SD.

DTT Reduction of Oxidized Troponin I

Troponin I that was incubated (air oxidized) at 1000 ng/ml in assaybuffer for 15 hours at room temperature was diluted to 4 and 8 ng/ml inassay buffer. DTT was added to a final concentration of 0, 1.5 and 3.0mM followed by incubation at room temperature for the times indicated inFIG. 2. The aliquots were then assayed for troponin I. After steadystate was reached (approximately 6 hours), aliquots (100 μl) from thethree DTT concentration samples were reoxidized with 20 mM peroxide for15 minutes, treated with catalase for 5 minutes and assayed. The resultsare shown in FIG. 2, wherein the error bars represent 1 SD.

The data show that the results of an immunoassay can vary over time ifthe oxidation-reduction state of the troponin I is allowed to change.The oxidation-reduction state of troponin I, and thus the immunoassayresults, can be reversibly changed and greatly stabilized over time bythe use of oxidants and reductants.

Example 5 Alkylation of Reduced Troponin I

Troponin I was rapidly alkylated using various alkylating reagents. Thestock reduced troponin I (University of Birmingham) was at 0.27 mg/ml in20 mM Tris hydrochloride, 0.5 M sodium chloride, 50 mM2-mercaptoethanol. Three alkylation reactions (#1-3) were performed anda control was prepared (#4):

1. 20 μl of stock troponin I was added to 20 μl 0.5 M potassium borate,0.2 mM ethylenediamine tetraacetic acid, pH 8.0 and subsequently, 10 μl398 mM iodoacetic acid was added.

2. 20 μl of stock troponin I was added to 20 μl 0.5 M potassium borate,0.2 mM ethylenediamine tetraacetic acid, pH 8.0 and subsequently, 10 μl398 mM iodoacetic acid was added.

3. 2 μl of stock troponin I was added to 20 μl 0.5 M potassium borate,0.2 mM ethylenediamine tetraacetic acid, pH 8.0 and subsequently, 12.5μl 319 mM N-ethylmaleimide was added.

4. 20 μl of stock troponin I was added to 20 μl 0.5 M potassium borate,0.2 mM ethylenediamine tetraacetic acid, pH 8.0 and subsequently, 10 μl0.5 M potassium borate, 0.2 mM ethylenediamine tetraacetic acid, pH 8.0was added.

The reactions were incubated at room temperature for 1 h 25 min. Duringthis incubation, the stock troponin I was kept on ice. Aliquots (24 μl)of each solution (1-4) were added to 0.9 μl of 2.9 M mercaptoethanol andwere incubated at room temperature for 15 min, after which the sampleswere frozen in liquid nitrogen. The remaining aliquots of each solution(1-4) were also frozen in liquid nitrogen with no further treatment.

Example 6 Immunoassay of Alkylated Troponin I

Freshly thawed Troponin I alkylated (Example 5) with N-ethyl maleimide(NEM), iodoacetic acid (IHAC), iodoacetamide (IAM), or not alkylated(control sample, Example 5) was diluted to 1-10 ng/ml in assay buffer. Afreshly thawed aliquot of the reduced stock troponin I (Example 5) wasdiluted (standard sample) into assay buffer containing either 0 or 3 mMDTT. Aliquots (25 μl) of all dilutions were taken after either a 0.5hour or 5.5 hour incubation at room temperature and assayed (Protocol A,Example 2) using a clone 2D5 anti troponin I monoclonal antibodyconjugate and biotinylated goat anti troponin I peptide 1 specificpolyclonal antibody (Example 1). This antibody pair hinds strongly tooxidized troponin I and weakly to reduced troponin I (Examples 3 and 4).Troponin I will remain substantially reduced during a 0.5 hourincubation but will be almost completely oxidized (by air) after a 5.5hour incubation unless DTT is present to stabilize the reduced form (seeExample 4). The results are shown in Table 2 and are given in terms ofassay slope (OD₄₉₀ per ng/ml total troponin I) in the linear range. Alarger assay slope indicates as stronger binding interaction between theantibodies and the troponin I.

The data show that the antibody pair binds alkylated troponin Isimilarly to reduced troponin I, that is, weakly in comparison withoxidized troponin I. Furthermore, alkylation stabilizes the immunoassayresult with respect to time, similarly to the effect observed by 0.25the use or oxidants or reductants to stabilize the oxidation-reductionstate of troponin I (Example 4). The lower and more stable assay slopeof the control sample as compared with the standard sample is explainedby the presence of mixed disulfides formed between the two cysteineresidues of the control sample troponin I and 2-mercaptoethanol duringthe room temperature incubation of the control sample at (see Example5).

TABLE 2 Assay slope Assay slope Ratio of assay (0.5 hour (5.5 hourslopes (5.5 hour/ Sample incubation) incubation) 0.5 hour) reduced TnI0.030 0.030 1.0 standard (+DTT) reduced TnI 0.058 0.21 3.6 standard(−DTT) TnI Control 0.037 0.078 2.1 TnI alkylated with NEM 0.023 0.0261.1 TnI alkylated with IAM 0.013 0.013 1.0 TnI alkylated with IHAC ≦0.01≦0.01

Example 7 Effect of Peroxide on Immunoassay of Cardiac Troponin I fromPatients with Confirmed Myocardial Infarction

Frozen Human serum or plasma, drawn in heparin tubes from patients withconfirmed myocardial infarction, was obtained from local hospitals. Theserum or plasma was thawed at room temperature and immediately splitinto two aliquots. One aliquot was oxidized at room temperature by theaddition of peroxide at a final concentration of 20 mM. The secondaliquot was untreated. The oxidation reaction was stopped after 20minutes by the addition of catalase at a final concentration of 0.01mg/ml. Ten minutes after the catalase was added both the oxidized andthe untreated aliquots were diluted serially by factors of four in assaybuffer and assayed immediately for cardiac troponin I using the 2D5 antitroponin I conjugate and biotinylated anti troponin I peptide 3 specificantibodies (Example 2, Protocol A). This complimentary antibody pairbinds oxidized troponin I strongly and reduced troponin I weakly(Example 3). Air oxidized (example 4) purified troponin I (P. Cummins,University of Birmingham), diluted to 2, 4, and 8 ng/ml in assay buffer,was assayed with the same antibody reagents to construct a standardcurve. The concentration of troponin I in the neat oxidized or untreatedserum or plasma sample (Table 3) was calculated from this standard curveusing the OD₄₉₀ measurements that fell within the linear range of theassay.

The data show that oxidation of serum or plasma samples from patientswith confirmed myocardial infarction can have a substantial effect onthe concentration of cardiac troponin I determined by immunoassay.Immunoassay of troponin I in serum or plasma without regard to theoxidation state of the troponin I could lead to a seriousunderestimation of the troponin I concentration and result in thenon-diagnosis of a myocardial infarction.

TABLE 3 Time between sample Troponin I concentration Troponin Iconcentration collection and by assay of untreated by assay of PeroxideSample freezing (hours) sample (ng/ml) oxidized sample (ng/ml) 1 Plasma2 6.3 9.4 2 Plasma 6.5 0.8 1.0 3 Serum 9.3 6.6 8.3 4 Serum 6.5 31.9 46.55 Plasma 6.5 31.7 49.7 6 Plasma 9.5 0.6 1.0 7 Serum 11.5 0.4 0.4 8Plasma 5.0 4.5 5.4 9 Serum 10.5 1.6 2.3 10 Plasma unknown 13.6 13.2 orSerum

Example 8 Effect of Peroxide on Immunoassay of Cardiac Troponin I inHuman Plasma Liter Two Freeze/Thaw Cycles

Plasma sample number 5 (Table 3, Example 7) was stored untreated on icefor three hours after it was initially thawed and then refrozen andstored at −70 C for several days. The plasma was thawed at roomtemperature and split into two aliquots; one was oxidized with peroxideand the oilier was left untreated as described in Example 7. Theconcentration of troponin I in the oxidized and untreated aliquots wasdetermined immediately by the immunoassay described in example 7 and wasfound to be 53.9 ng/ml in the untreated aliquot and 56.4 ng/ml in theoxidized aliquot.

The data show that oxidation of the plasma after the second thaw did nothave a substantial effect on the concentration of cardiac troponin Idetermined by immunoassay.

Example 9 Immunoassay of Cardiac Troponin I in Oxidized and ReducedPlasma from a Patient With Myocardial Infarction

Frozen Human plasma drawn in heparin tubes from a patient with aconfirmed myocardial infarction was obtained from a local hospital. Theplasma was thawed at room temperature and immediately split into twoaliquots. One aliquot was oxidized with peroxide as described in Example7. The other aliquot was reduced by addition of DTT to a finalconcentration of 10 M, followed by a 3 hour incubation at roomtemperature. The oxidized aliquot was then diluted serially by factorsof 2 into assay buffer and the reduced aliquot was diluted serially byfactors of 2 into assay buffer containing 3 mM DTT. The diluted aliquotswere assayed for troponin I immediately (Protocol A, Example 2) eitherwith the complementary antibody pair clone 2D5 anti troponin I conjugateand biotinylated anti troponin I peptide 3 polyclonal antibody or withthe complementary antibody pair clone TRI-7 F81 anti troponin Iconjugate and biotinylated anti troponin I peptide 3 polyclonalantibody. Purified troponin I (University of Birmingham) which was airoxidized (example 4) was diluted to 2, 4, and 8 ng/ml in assay bufferand was used to construct the standard curve from which theconcentration of troponin I in the neat oxidized or reduced plasmasample was determined. The results are shown in Table 4.

The data show that chemical oxidation and reduction of cardiac troponinI in the plasma sample affects the recognition of the tested antibodypairs for the troponin I in a manner similar to that observed forpurified troponin I (Example 3).

TABLE 4 Monoclonal Assayed troponin I Assayed troponin I Ratio oftroponin I antibody concentration (ng/ml) in concentration (ng/ml)concentrations (oxidized conjugate oxidized plasma in reduced plasmaplasma/reduced plasma) Clone 2D5 82 <1 >82 Clone TRI-7 F81 52.8 77.50.68

Example 10 Selection of Anti Troponin I Antibodies that are EitherSensitive or Insensitive to the Binding of Troponin C to Troponin I

Monoclonal anti troponin I conjugates and complimentary biotinylatedanti troponin I polyclonal antibodies (Example 1) were tested for theirrecognition of free troponin I and troponin I bound to troponin C in abinary complex. Four types of troponin I samples were prepared at roomtemperature and assayed for troponin I; they are: oxidized troponin Iwith and without added troponin C and reduced troponin I with andwithout added troponin C. Oxidized (by air, see Example human cardiactroponin I (P. Cummins, University of Birmingham) was diluted to 2, 4,and 8 ng/ml in assay buffer containing 2 mM calcium chloride. Onealiquot of each concentration of troponin I was either untreated orreduced by the addition of DTT to a final concentration of 3 n110 from a30 mM DTT stock solution in assay buffer to form a reduction reaction.Three hours after the reduction reaction was started, each oxidized andreduced troponin I aliquot was split into two aliquots; to one aliquotwas added human cardiac troponin C (Bio-Tech International Inc.,Seattle, Wash.) to a final concentration of 0.1 mg/ml from al mg/mlstock solution in 20 mM potassium phosphate, 4 mM potassium borate, 150mM sodium chloride, pH 7.0 to form a binding reaction mixture, and tothe other aliquot was added the same volume of the above buffer withouttroponin C. One hour after the troponin C was added, all the aliquotswere assayed for troponin I (Protocol A, Example 2) using the antibodypairs listed in Table 5. The results in Table 5 are expressed as afractional assay response which was determined by dividing the assayslope in the presence of troponin C by the assay slope in the absence oftroponin C.

The results in Table 5 show that some antibody pairs recognize freetroponin I and troponin I bound to troponin C equally well, while otherantibody pairs recognize only tree troponin I. An immunoassay withantibodies that do not recognize troponin I bound to troponin C willunderestimate the total troponin I concentration when some of thetroponin I is present as the troponin I/C binary complex.

TABLE 5 Biotylated Fractional assay response Anti troponin I antitroponin I Oxidized Reduced antibody conjugate polyclonal antibodytroponin I troponin I Clone 2D5 Peptide 3 0.81 0.60 specific Clone 111Peptide 1 0.83 Not specific determined Clone 111 Peptide 3 0.47 0.52specific Clone 111 Peptide 4 0.59 0.19 specific Clone 110 Peptide 4 0.960.48 specific Clone 1A12 Peptide 1 <0.05 <0.05 specific Clone 1A12Peptide 3 <0.05 <0.05 specific Clone 1A12 Peptide 4 <0.05 <0.05 specificClone TR7 F81 Peptide 1 0.74 0.79 specific Clone TR7 F81 Peptide 2 0.921.04 specific Clone TR7 F81 Peptide 3 0.94 0.97 specific Clone TR7 F81Peptide 3 0.70 0.79 specific

Example 11 Effect of Troponin EDTA, Melittin and Mastoparan on aTroponin I Immunoassay with Troponin C Present in Large Excess OverTroponin I

Ethylenediamine tetraacetic acid (EDTA) lowers the binding affinity oftroponin | for troponin I and troponin C by chelating calcium andmagnesium ions. Melittin lowers the affinity of troponin I for troponinC by binding to troponin C. The effectiveness of EDTA and Melittin(hereafter referred to as binding inhibitors) in breaking up the binarycomplex of troponin I and troponin C in the presence and absence oftroponin T was assessed. Oxidized Human cardiac troponin I (P. Cummins,University of Birmingham) at 1.0 ug/ml in assay buffer containing 2 mMcalcium chloride was reduced with dithiothreitol at a finalconcentration of 3 mM for three hours at room temperature. The reducedtroponin I was diluted to 2 and 4 ng/ml in assay buffer containing 2 mMcalcium chloride and 3 mM dithiothreitol. Each concentration was splitinto four aliquots to which were added human cardiac troponin C(Bio-tech International, Inc.) to final concentrations of 0, 0.1, 1.0,and 10.0 μg/ml from 100-fold excess stock solutions in 20 mM potassiumphosphate, 4 mM potassium borate, 150 mM sodium chloride, pH 7.0. Eachof the resulting aliquots were further split into two aliquots to whichwas added human troponin T (Scripps Labs) to a final concentration ofeither 0.0 or 0.1 μg/ml from a 100-fold excess stock solution deionizedwater. The aliquots were incubated at room temperature for one hourafter the addition of troponin T, then assayed for troponin I (ProtocolB, Example 2). The antibody solution added to the microtiter plate wellscontained 30 μg/ml clone 1A12 anti troponin 1 and 7.5 μg/ml biotinylatedanti troponin I peptide 4 specific antibodies (example 1) either withoutor with binding inhibitors (30 mM EDTA and 0.15 mM Melittin (SigmaChemical, Co., St. Louis, Mo.)). Aliquots of the reaction mixturesformed by the addition of antibodies to the troponin I samples wereremoved after 0.5 bond were further treated as described in Protocol B,Example 2. The assay results for the samples containing no troponin C orT and no binding inhibitors were used to construct a standarddose-response curve. The effect on the standard curve of addition of thebinding inhibitors to the assay was tested and found to be negligible.The fractional assay response (shown in FIG. 3) for samples containinginhibitors and troponin components was determined by dividing the assayslope for each sample by the slope of the standard curve.

The data show that in the presence of troponin C, the troponin Iconcentration is largely underestimated. The binding inhibitors almostcompletely reverse the effect of troponin C. The presence of troponin Tin the absence of troponin C has no effect on the troponin Iimmunoassay. (Data not shown in FIG. 3). In the presence of troponin Cand T, the measured concentration of troponin I is dramatically reduced.The binding inhibitors appear to be less effective at opening up orpartially unraveling the troponin complex when the complex is ternarythan when the complex is binary. Mastoparan or Melittin at 0.1 mM wasalso tested as a binding inhibitor to dissociate the I/C complex for atroponin C concentration of 10 ug/ml (Data not shown). The melittin wasas effective as the melittin/EDTA (FIG. 3) at increasing the fractionalassay response, while the mastoparan was about one third as effective asthe melittin at the concentrations tested.

Example 12 Effect of Binding Inhibitors on an Immunoassay of Troponin Iin the Presence of Troponin C or Troponin C and T

Solutions containing 1.0 μg/ml purified human cardiac troponin I(reduced by DTT, Example 4) and either 1.2 μg/ml human cardiac troponinC (Bio-tech International, Inc.) or 1.2 μg/ml troponin C and 3.1 μg/mlhuman cardiac troponin T (Scripps Labs) were incubated for 2 hours atroom temperature in assay buffer containing 3 mM DTT. Troponin C wasadded to troponin I prior to addition of troponin T. The troponinsolutions were diluted to 2.4 and 8 ng/ml in terms of troponin Iconcentration in assay buffer containing 2 mM calcium chloride and 0.5mM DTT and assayed immediately with and without binding inhibitors asdescribed in example 11. Aliquots of the reaction mixtures of antibodiesand troponin components were removed 0.5 h and 2.2 h after theantibodies were added. These aliquots were further treated as describedin Protocol B, Example 2. Reduced troponin I without added troponin Cand T and without binding inhibitors was assayed to produce a standardcurve. The effect on the standard curve of addition of the bindinginhibitors to the assay was tested and found to be negligible. Theresults are expressed in Table 6 as a fraction assay response which wasdetermined by dividing the assay slope for each sample by the slope ofthe standard curve.

The data show that troponin T and C present in approximately a two foldmolar excess above the troponin I concentration substantially towers theamount of troponin I measured in the immunoassay. Troponin C alone has asmaller effect on the measured troponin I concentration at the antibodyconcentrations used in this assay. The binding inhibitors partiallyreverse the effect of troponin C and T at 0.5 h incubation andcompletely at 2.2 h.

TABLE 6 Fractional assay response With troponin C With troponin C and TTime after without with without with antibodies added binding bindingbinding binding (hours) inhibitors inhibitors inhibitors inhibitors 0.50.88 0.94 0.55 0.75 2.2 1.15 1.02 0.44 1.02

Example 13 Assay of Purified Human Cardiac Ternary Troponin Complex forTroponin I

Purified human cardiac ternary troponin complex (Bio-tech International,Inc., 3 mg/ml stock solution in a buffer containing 20 mM sodiumcitrate, pH6) was diluted to concentrations ranging from 1 to 15 ng/mltotal troponin I in assay buffer containing 2 mM calcium chloride butwithout the 0.1 mM zinc chloride or the 1 mM magnesium chloride(hereafter called metal-free assay buffer). The diluted solutions wereassayed for troponin I with and without binding inhibitors present asdescribed in Example 11. Aliquots of the reaction mixtures of theantibodies with the troponin complex were taken at the times indicatedin FIG. 4 and were further treated as described in Protocol B. Example2. Purified troponin I (Bio-tech International, Inc.) was assayed andused to construct a standard curve. The effect on the standard curve ofaddition of the binding, inhibitors to the assay was tested and found tobe negligible. The fractional assay response shown in FIG. 4 wasdetermined by dividing the assay slope for the complex (OD₄₉₀ as afunction or total troponin I concentration) by the slope of the standardcurve.

The results show that troponin I that is bound in the ternary complexwith troponin C and T is not recognized very well by the antibodies atthe antibody and troponin I concentrations used in this assay, evenafter a very long incubation time. In particular, the 30 minute timepoint had no detectable troponin I, with or without inhibitors. Thebinding inhibitors slowly open up or partially unravel the complex andthus slowly increase the fraction of troponin I recognized by theantibodies.

Example 14 Effect of Binding Inhibitors on a Troponin I Immunoassay ofPlasma and Scrum from Patients with Confirmed Myocardial Infarction

Frozen serum and plasma drawn in EDTA and Heparin tubes from patientswith a confirmed myocardial infarction were obtained from localhospitals and thawed at room temperature. Calcium chloride was added toa final concentration of 6 mM (to bind all the EDTA) and the resultantsolution was incubated for two hours at room temperature and thenovernight at 4 C. The incubated samples were diluted by factors or twoto a maximum dilution factor of 256 in metal-free assay buffer. Thediluted samples were immediately assayed for troponin I with and withoutbinding inhibitors as described in Example 11, with the exception thatthe polyclonal antibody was goat anti-troponin I peptide 3 specific.Aliquots of the reaction mixture formed by the addition of antibodies tothe diluted samples were taken at the times indicated in FIGS. 5 a-f andfurther treated as described in Example 2, Protocol B. Oxidized troponinI (University of Birmingham) at 2, 4 and 8 ng/ml in metal-free assaybuffer was assayed to produce a standard curve. The effect on thestandard curve of addition of the binding inhibitors to the assay witstested and found to be negligible. The OD₄₉₀ values measured for thediluted serum or plasma samples were plotted as a function of theinverse of the dilution factor. The slope in the linear region of theresultant curve (typically at OD₄₉₀<2, which corresponds to a troponin Iconcentration of less than 8 ng/ml) was divided by the slope of thestandard curve to obtain the concentration of troponin I in the neatserum or plasma sample shown in FIGS. 5 a-f. Each FIG. 5 a through 5 freflects immunoassays on serum or plasma from different patients.

The data show that the measured concentration of troponin I in all ofthe serum and plasma samples tested was increased by the addition ofbinding inhibitors. Importantly, the time profile of the measuredconcentration of troponin I was in some cases biphasic (FIGS. 5 a-c and5 e). The fast phase was complete within the first assay time of 0.5 h.The slow phase continued to rise for 6-24 hours depending on the sample.A slow phase was also observed for the purified ternary troponin complex(Example 13). The slow phase observed for the diluted serum and plasmasamples, may, therefore, be associated with the opening up or partialunraveling of a ternary complex by the inhibitors and antibodies. Thefast phase indicates a bound complex of troponin I that is more easilyopened up or partially unraveled than the ternary complex, since thefast phase is absent for the purified ternary complex (Example 13).Thus, the fast phase could be associated with the opening up or partialunraveling of binary complexes of troponin I.

Example 15 Immunoassays that are Sensitive to Free Troponin I, TroponinI Bound in a Ternary Complex and Both Free and Bound Troponin I

Three sets of antibodies were evaluated for their ability to recognizefree troponin I and troponin I bound in the ternary complex. Threeantibody stock solutions described below as #1-3 were prepared inmetal-free assay buffer either with or without binding inhibitors (30 mMEDTA and 0.15 mM Melittin) and the following antibodies:

1. 30 μg/ml 1A12 anti troponin I and 7.5 μg/ml biotinylated antitroponin I peptide 4 specific;

2. 30 μg/ml 1A12 anti troponin I, 30 μg/ml anti troponin T monoclonal(Biospacific), 5 μg/ml each of biotinylated anti troponin I peptide 1,2, 3 and 4 specific;

3. 30 μg/ml 9B1 anti troponin T and 5 μg/ml each of biotinylatedbiotinylated anti troponin I-peptide 1, 2, 3 and 4 specific.

Human cardiac troponin ternary complex (Bio-tech International, Inc.)was diluted to 1-15 ng/ml troponin I equivalents in metal free assaybuffer and purified troponin I (Bio-tech International, Inc.) wasdiluted to 2, 4 and 8 ng/ml in the same buffer. The dilutions of thetroponin complex and troponin I were assayed immediately using ProtocolB, Example 2 with antibody solutions #1-3. Aliquots of the reactionmixtures formed by the addition of the antibodies to the troponincomplex and troponin I samples were taken at 0.5 h and 2.5 h after theantibodies were added and were further treated as described in Example2, Protocol B. The results are shown in Table 7 and are expressed interms of an ASSAY slope with units of OD₄₉₀ per ng/ml total troponin Iin the linear range of the assay. A higher slope indicates betterbinding of the antibodies to the troponin components. Antibody solution#2 was tested and found to be negative for cross reactivity withpurified human cardiac troponin T (Scripps Labs) at 1-6 ng/ml using theassay protocol described herein (data not shown).

The data show that the immunoassay using the antibodies in solution #1recognizes free troponin I but not troponin I in the ternary complex.The immunoassays using the antibodies in Solution #2 recognizes freetroponin I and troponin I in the ternary complex almost equally well.Thus, antibody solution #2 is superior to solution #1 for the assay oftotal troponin I when a fraction of the troponin I is present as theternary complex. The immunoassay using the antibodies in solution #3recognizes the ternary complex well but recognizes free troponin Ipoorly. This poor recognition of free troponin I causes the assay slopefor antibody solution #3 to decrease over time in the presence ofbinding inhibitors. By using all three antibody solutions inimmunoassays, one can estimate independently the concentrations of freetroponin I (solution #1), total troponin I (solution #2) and boundtroponin I (solution #3).

TABLE 7 Assay Slope (OD₄₉₀ per ng/ml total troponin I) Time afterTroponin ternary Troponin ternary Antibody antibodies complex (withoutcomplex (with solution added Free binding binding # (hours) Troponin Iinhibitors) inhibitors) #1 0.5 0.14 <0.006 <0.006 #1 2.5 0.14 0.0030.021 #2 0.5 0.12 0.18 0.14 #2 2.5 0.22 0.17 0.12 #3 0.5 0.008 0.18 0.17#3 2.5 0.008 0.17 0.08

Example 16 Estimation of Free Troponin I, Bound Troponin I and TotalTroponin I in Plasma from a Patient with a Myocardial Infarction

The three antibody solutions described in Example 15 were used inimmunoassays to measure the troponin I concentration in plasma from apatient with at confirmed myocardial infarction. The frozen plasma wastreated and diluted into metal free assay buffer as described in Example14. The diluted plasma was assayed for troponin I immediately usingProtocol B. Example 2 with antibody solutions #1-3 (Example 15).Aliquots were taken 0.5 h and 2.5 h after the antibodies were added tothe samples to form the reaction mixtures and were further treated asdescribed in Example 2, Protocol B. Either free troponin I at 1-4 ng/mlor the troponin complex (Bio-tech International, Inc.) at 1-8 ng/mltroponin I in metal-free assay buffer was assayed and used to constructa standard curve of OD₄₉₀ as a function of total troponin Iconcentration for each antibody solution. The concentration of troponinI in the neat plasma sample, as shown in Table 8, measured by eachantibody solution was determined using either the standard curve forfree troponin I or for troponin I in the ternary complex in the absenceof binding inhibitors as indicated in Table 8 and as described inExample 14. The ratio determined by dividing the assayed troponin Iconcentration with binding inhibitors by the concentration withoutinhibitors is also shown in Table 8.

The data show that the concentration of troponin I determined byimmunoassay using antibody solution #1 is much more sensitive to thepresence of binding inhibitors and thereby to the opening up or partialunraveling of the troponin complex than that determined using solution#2 or #3. The data of Example 15 suggest that antibody solution #1measures mainly free troponin I, solution #2 measures both free troponinI and troponin I bound in the ternary complex and solution #3 measuresmainly troponin I bound in the ternary complex. Thus, the conclusionsfrom Example 15 taken together with the data in Table 8, indicate thatsubstantial amounts of both free and bound troponin I are present in thediluted plasma sample. Among the three antibody solutions used in theimmunoassays, solution #2 gives the largest assayed troponin Iconcentration, as expected, because the immunoassay using antibodysolution #2 measures both free and bound troponin I. Thus, antibodysolution #2 appears to provide the most comprehensive measure oftroponin I in the plasma sample. Antibody solution #2 standardized withthe purified troponin complex gave the most stable assay with respect toinhibitor addition and assay incubation time. The decrease of troponin Iconcentration at 2.5 h measured with antibody solution #2 standardizedwith free Troponin I is due to an increase at 2.5 h of the slope of thestandard curve.

TABLE 8 Troponin I concentration in Time after Standard used plasma,(ng/ml) antibodies to determine Without With Antibody added, troponin Ibinding binding solution (hours) concentration inhibitors inhibitorsRatio #1 0.5 Troponin I 91 186 2.0 #1 2.5 Troponin I 83 251 3.0 #3 0.5Ternary 134 108 0.8 Troponin Complex #3 2.5 Ternary 87 65 0.74 TroponinComplex #2 0.5 Troponin I 280 360 1.3 #2 2.5 Troponin I 172 220 1.3 #20.5 Ternary 229 299 1.3 Troponin Complex #2 2.5 Ternary 223 286 1.3Troponin Complex

Example 17 Immunoassay of Free Human Cardiac Troponin T and Troponin Tin the Human Cardiac Ternary Complex

Two antibody stock solutions (#1 and 2) were prepared as described belowin metal-free assay buffer either with or without binding inhibitors (30mM EDTA and 0.15 mM Melittin) and with the following antibodies:

1. 30 μg/ml 1A12 anti troponin I, 30 μg/ml 9B1 anti troponin T and 7.5μg/ml biotinylated anti troponin T peptide 3 specific (Biospacific).

2. 30 μg/ml 9B1 anti troponin T and 7.5 μg/ml biotinylated anti troponinI peptide 3 specific.

Human cardiac troponin ternary complex (Bio-tech International, Inc.)was diluted to 1-20 ng/ml troponin T in metal free assay buffer andpurified Human cardiac troponin T (Scripps Labs) was diluted to 1.5, 3.0and 6.0 ng/ml in the same buffer. The dilutions of the troponin complexand troponin T were assayed immediately using Protocol B, Example 2 withantibody solutions #1 and #2. Aliquots of the reaction mixtures formedby the addition of the antibodies to the troponin complex and troponin Tsamples were taken at 0.5 h and 3.0 h after the antibodies were addedand were further treated as described in Example 2, Protocol B. Theresults are shown in Table 9 and are expressed in terms of an assayslope with units of OD₄₉₀ per ng/ml total troponin T in the linear rangeof the assay. A higher slope indicates better binding of the antibodiesto the troponin components.

The data show that the antibodies in solution #1 recognize both freetroponin T and troponin T bound in the ternary complex equally well. Theantibodies in solution #2 recognize free troponin T well but recognizestroponin T in the ternary complex poorly. Thus, antibody solution #1 isexpected to provide the most comprehensive and accurate measure of thetotal concentration of troponin T in human blood samples in which asubstantial amount of the ternary complex is present.

TABLE 9 Time after Assay slope (OD₄₉₀ per ng/ml troponin T) antibodiesTroponin Troponin added to Free troponin T Free troponin T complexcomplex with Antibody troponin sample without binding with bindingwithout binding binding Solution (hours) inhibitors inhibitorsinhibitors inhibitor #1 0.5 0.069 0.078 0.061 0.070 #2 0.5 0.078 0.0850.013 0.012 #2 3.0 >0.08 >0.08 0.018 0.023

Example 18 Use of Troponin C to Prevent Non-Specific Binding of TroponinI to Filter Membranes

Oxidized cardiac troponin I (air oxidized, Example 4, University ofBirmingham) at 100 ng/ml final concentration in human serum (Hybritech,Inc., San Diego) was incubated either with or without 100 ug/ml humancardiac troponin C (Bio-tech International, Inc.) at room temperaturefor 30 minutes. Two filter membranes, a CytoSep filter (AhlstromFiltration, Mount Holly Springs, Pa.) and a glass fiber filter (GB-100R,Micro Filtration Systems, Dublin, Calif.) were cut into rectanglesmeasuring 1.5 cm by 3.0 cm and were secured to a transparency film(catalog #pp 2500, 3M, Austin, Tex.) by a piece of tape across thefilters. The troponin I solutions with and without troponin C (300 μl)were applied slowly to the top of the filters at one end and thesolution migrated through, the filter to the far end by wicking action.About 15 μl of solution was collected from the far end with a plasticpipet tip. The collected solutions were diluted by factors of 20, 40 and80 in assay buffer and assayed using Protocol A, Example 2 with TRI-7F81 anti troponin I conjugate and biotinylated anti troponin I peptide 3specific antibodies. Aliquots of the troponin I solutions with andwithout troponin C that had not been passed through the filters werealso assayed and used to construct a standard curve from which theconcentration of troponin I in the solution that had passed through themembrane was determined. The calculated concentration was divided by 100ng/ml to obtain the fraction of recovered troponin I shown in Table 10.The experimental errors given in Table 10 represent one standarddeviation.

The data show that the presence of troponin C helps to lower thenon-specific binding at troponin I to the filter membranes.

TABLE 10 Fraction of troponin I recovered Filter Without troponin C Withtroponin C CytoSep 0.03 ± 0.03 0.15 ± 0.04 Glass Fiber 0.00 ± 0.03 0.09± 0.04

Example 19 Use of Proteins of High Isoelectric Point to PreventNon-Specific Binding of Troponin I to Filter Membranes

A blood filter (CytoSep 1.5 cm×3.0 cm) was soaked for 16 hours at roomtemperature in solutions of deionized water containing 1 mg/ml of theproteins listed in Table 11. The filters were rinsed once with deionizedwater and dried for 2 hours at 35 C. Oxidized human cardiac troponin I(Bio-tech International, Inc.) at 100 ng/ml in human serum (Hybritech,Inc., San Diego, Calif.) also containing 2 mM added calcium chloride waspassed through the filters as described, in Example 18. The amount oftroponin I recovered from the filters was determined by assay (ProtocolB, Example 2) using TRI-7 F81 anti-troponin I and biotinylated antitroponin I peptide 4 specific antibodies with added binding inhibitors(Example 11). The data in Table 11 are expressed as the fraction oftroponin I recovered, which was determined as described in Example 18.

The results show that Melittin and protamine sulfate substantiallyreduces the non-specific binding of troponin I to the blood filter,whereas casein and non-fit dried milk had little effect at theconcentrations tested.

TABLE 11 Fraction of troponin I Protein recovered No addition 0.16Protaraine Sulfate 0.77 Casein 0.16 Melittin 0.72 Non-fat dried milk0.08

Example 20 Immunoassay of Ternary Troponin Complex Using Tri-7 F81 AntiTroponin I and Biotinylated Anti Troponin I Peptide 4 Antibodies

The purified ternary troponin complex (Bio-tech International, Inc.) wasassayed for troponin I as describe in Example 13, except that the titleantibody pair was used in the immunoassay and the aliquot of thereaction mixture of the antibodies with the troponin sample was takenthree hours after the antibodies were added to the troponin. Thefractional assay response was 0.16 in the absence of binding inhibitorsand 0.49 in the presence of binding inhibitors.

The data show that the title antibody pair recognizes troponin I in theternary complex poorly. In example 10, it was shown that the presence oftroponin C without Troponin I had little effect on the recognition ofthe title antibody pair for to troponin I. Thus, the title antibody paircan bind to troponin I present in the binary complex with troponin Cbetter than it can bind to Troponin I present in the ternary complex.

Example 21 Immunoassay of Troponin I in Plasma from a Patient withConfirmed Myocardial Infarction Using TRI-7 F81 Anti Troponin IConjugate and Biotinylated Anti Troponin I Peptide 1 Specific Antibodies

Frozen plasma from a patient with a continued myocardial infarction wasthawed and diluted in human serum (Hybritech Inc., San Diego, Calif.)also containing 0.5 M added sodium chloride and assayed for troponin Iwith the title antibody pair using Protocol A, Example 2 oxidizedpurified human cardiac troponin I (University of Birmingham) was assayedand used to construct a standard curve from which the troponin I in theplasma was determined. The neat plasma sample was refrozen in a −70 Cfreezer after being on ice for several hours. The frozen plasma wasrethawed at room temperature, and was assayed using the same protocol asdescribed above except the plasma and troponin I standards were dilutedinto assay buffer. The standards exhibited almost identical assay slopeswhen diluted in serum (first assay) or assay buffer (second assay). Theneat plasma sample was further incubated at 4 C for the times indicatedin Table 12 and reassayed in assay buffer.

The data show a substantial increase in the assayed troponin Iconcentration after a freeze/thaw cycle and after incubation at 4 C.This assay instability may be associated with the opening up or partialunraveling of the ternary troponin complex by the freeze/thaw cycle.

TABLE 12 Assayed concentration Time of assay of troponin I After firstthaw of plasma  284 ng/ml  2 hours after second thaw of plasma >800ng/ml 19 hours after second thaw of plasma 1760 ng/ml 90 hours aftersecond thaw of plasma 2300 ng/ml

Example 22 Expression, Screening and Selection of Recombinant AntibodiesBinding Fragments or Fab Fragments

Immunization of Mice

Mice were immunized by the following method. Each of ten mice wereimmunized intraperitoneally using 50 μg protein antigen (troponin I andtroponin ternary complex) emulsified in Freund's complete adjuvant onday 0, and day, 28. Test bleeds of mice were obtained through punctureof the retro-orbital sinus. When the titers were determined to be highfor biotinylated antigen, the mice were boosted with 50 μg of protein onday 70, 71, and 72, with subsequent sacrifice and splenectomy on day 77.If titers of antibody were not satisfactory, mice were boosted with 50μg antigen on day 56 and a test bleed was obtained on day 63. Ifsatisfactory titers were obtained the animals were boosted with 50 μg ofantigen on day 98, 99, and 100 and the spleens extracted on day 105.

Antibody Phage Display Libraries

Mice having high titers of antibodies to the desired protein antigen(troponin I or troponin ternary complex) were sacrificed, and total RNAwas purified from the spleen cells (Anal. Biochem, 162: 156-159 (1987)).The total RNA (50 μg) from the spleen cells was used directly astemplate for Superscript™ II reverse transcriptase (Gibco BRL,Gaithersburg, Md.) with oligo (dT)₁₂ as the primer to make cDNA for PCR.A total of 32 PCR reactions was used to amplify the heavy chain variableregions using Tag DNA polymerase (Boehringer Mannheim, Indianapolis,Ind.), 3 μL cDNA per reaction, 32 different 5′ oligonucleotides and assingle 3′ oligonucleotide. The kappa chain variable regions wereamplified similarly using 29 different 5′ oligonucleotides and a single3′ oligonucleotide. The oligonucleotides were previously designed toensure that nearly all antibody variable regions present in the spleenwould be amplified with no more than 2 changes in the actual amino acidsequence caused by mismatches of the oligonucleotide with the cDNA basedon a compilation of mouse antibody sequences. (Sequences of proteins ofimmunological interest. Vol. 2, 5th edition, 1991, Cambridge, Mass.) Analiquot of each double stranded PCR product was amplified a second timeusing only the 3′ oligonucleotide to produce single stranded DNA(ss-DNA). The ss-DNA products from all of the kappa chain amplificationreactions were pooled, and the ss-DNA products from all of the heavychain reactions were pooled separately. The ss-DNA from each pooledsample was purified using high performance liquid chromatography (HPLC)and a Genpak Fax HPLC column (Waters Inc, Milford, Mass.). The purifiedss-DNA was used to perform mutagenesis of an M13 uracil templatefollowing standard oligonucleotide directed mutagenesis techniques (J.Immunol. 149: 3903-3913, 1992). The M13 template contains DNA sequencescomplementary to the 5′ and 3′ ends of the ss-DNA for both the heavychain and the kappa chain variable regions. As a result, when the heavychain and kappa chain variable regions were annealed to the M13 vector,a mixture of different antibody sequences was created. The resultingantibodies were expressed as Fab antibody fragments comprising theentire kappa chain and the variable region of the heavy chain linked tothe first constant, region of the heavy chain. Electrocompetent cells(DH12S Gibco BRL., Gaithersburg, Md.) were transformed byelectroporation with the resultant M13 DNA. A typical electroporation of500 ng M13 vector into 40 μL DH12S cells yielded 10⁷ to 10⁸ differentantibody clones. The clones were further amplified to produce anantibody phage display library. The M13 vector was designed so that theheavy chain was expressed as a fusion protein with the major coatprotein of M13, the gene VIII protein. There are approximately 2700copies of the gene VIII protein of E. coli, phage particle. After theheavy chain/gene VIII fusion protein was secreted to the periplasm of E.coli, the secreted kappa chain binds to the heavy chain to form afunctional antibody Fab fragment. This leads to the production of phagehaving functional antibodies on the surface of the phage with the DNAencoding the antibody packaged inside the phage.

Screening of Antibody Phage Display Libraries

The antibody phage display library obtained from electroporating the M13mutagenesis DNA into E. coli consists of many different antibodiesdisplayed on phage particles (antibody phage). The number of highaffinity antibodies to the protein antigen is low. The followingscreening procedure was developed to isolate antibodies specific totroponin I and the troponin ternary complex. Antibody phage wasincubated with biotinylated, oxidized troponin I (10⁻⁹M, 10biotins/protein) or biotinylated troponin ternary complex (10⁻⁹M, 11biotins/protein) in solution to equilibrium. The antibody phage bindingto biotinylated protein antigen was isolated by incubation with magneticlatex coated with avidin. The nonspecific antibody phage were washedaway, and antibody phage binding to latex was eluted and amplified bygrowth in XL1 blue E. coli cells (Stratagene, La Jolla, Calif.). Theamplified antibody phage were then subjected to a second round ofselection. Since the incubation of biotinylated protein antigen andantibody phage was performed in solution, the concentration ofbiotinylated protein antigen was adjusted to select only for highaffinity antibodies. The process described above was repeated until theantibody phage consisted of a high percentage of phage encoding troponinantibodies. The antibody sequences were then ready for subcloning.

Antibody Subcloning

The entire antibody DNA sequence was amplified using a 5′oligonucleotide binding to the signal sequence of the kappa chain, whichis on the 5′ side of the heavy chain, and a 3′ oligonucleotide bindingto the end of the heavy chain constant region sequence. Afteramplification, the DNA was purified using agarose gel electrophoresis,then annealed and ligated to a pBR322 expression vector. Transformationof DH10B cells by electroporation was accomplished using this DNA, andthe cells were grown on tetracycline plates to select clones containingthe inserted DNA. Colonies were transferred into 3 mL 2YT media with 10μg/mL tetracycline, and cultures were grown overnight at 37; C.Overnight cultures of cells were diluted 1:100 into 50 mL, 2YT mediawith 1% glycerol and 10 mg/mL, tetracycline in 250 mL baffled shakeflasks to obtain sufficient antibody for testing. Cultures were grown at37; C until the absorbance of the culture at 600 nm was between 2 and 4.At that point, cultures were induced and grown overnight at 23; C. Thecells were disrupted in a high pressure homogenizer, and the antibodywas purified.

Antibody Characterization by Epitope Mapping

The antibodies produced were characterized with regard to their abilityto perform in a double antibody, solid-phase, noncompetitive enzymeimmunoassay (sandwich assay). Antibody Fab fragments (Fab) were selectedto bind distinctly different epitopes on the protein antigen so that thebinding of one Fab did not sterically hinder the binding of the secondFab. The protein antigen was labeled with biotin by using biotin-XX-NHSester (Molecular Probes, Portland, Oreg.). The biotinylation of theprotein antigen was sufficiently low in molar ratio of biotin to protein(on average, 4 biotin protein) so that the probability of an epitopebeing substantially affected with regard to the native structure of theantibody binding site was minimal. The first Fab fragment was incubatedin microtiter plate wells which contained adsorbed, affinity-purifiedgoat-anti-mouse Fab. The wells were washed and a blocking solutioncontaining a non-specific mouse Fab at high concentrations was placed ineach well to saturate remaining anti-Fab sites. In separate microtiterplate wells, the second Fab fragment was incubated to equilibrium withthe biotinylated protein antigen. The second Fab was present insubstantial molar excess over the biotinylated protein antigen. Themixture containing the second Fab and the biotinylated protein was addedto the microliter plate wells that were coated with the first lab andincubated. A conjugate of streptavidin and alkaline phosphatase wasadded to the mixture to bind to the biotinylated protein to detect thepresence of the protein antigen. The microtiter wells were washed andthe presence of bound alkaline phosphatase was detected by addingphenolphthalein monophosphate, and the rate of product formation at 560nm was determined using a spectrophotometric microliter plate reader.The excess non-specific mouse lab in the mixture prevented the secondFab from binding to anti-Fab adsorbed to the wells of the microtiterplate. Controls were performed using the same lab as the first andsecond Fab to show that when the epitope bound by the second Fab is thesame as the first very little binding of the alkaline phosphatase to themicrotiter plate well was observed, if the two different Fab fragmentsbeing tested can bind to different epitopes on the same protein, thenthe amount of alkaline phosphatase activity bound to the well wassubstantially greater than that of the control. The advantage of thisassay procedure is that the antibodies were unlabeled so that manyantibodies can be rapidly assayed to determine if they bind distinctlydifferent epitopes. The number of distinct epitopes was determined andantibodies were grouped according to their epitope specificity.

Example 23 Characterization and Selection of Complementary AntibodyPairs for Recognition of Oxidized and Reduced Free Troponin I andTroponin I Bound in the Ternary Complex

Antibodies directed to troponin I were tested in a sandwich immunoassayin pairs consisting of an antibody conjugate antibody and acomplementary biotinylated antibody (example 1). The antibodies testedwere anti troponin I recombinant antibodies that were originally raisedto free troponin I but were selected to bind both free troponin I andtroponin I in the ternary complex (example 22). Goat anti troponin Ipeptide 3 specific polyclonal antibody was also tested.

Purified human cardiac ternary troponin complex (Bio-Tech International)was diluted into assay buffer and assayed (protocol A, Example 2) withthe complementary antibody pairs shown in Table 13. The results areexpressed as an assay slope with units of OD₄₉₀ per ng ml total troponinI in the linear range of the assay. A higher slope indicates betterbinding of the antibodies to the troponin I.

Troponin I was prepared by dissociating the components of the ternarytroponin complex under oxidizing (hereafter called dissociated/oxidizedsample) and reducing (hereafter called dissociated/reduced sample)conditions. Purified human cardiac ternary troponin complex wasincubated at 4 ug/ml for 4 hours at room temperature in a dissociationbuffer consisting of 50 mM 3-(N-morpholino) propane sulfonic acid, 150mM sodium chloride, 6 M urea, 10 mM EDTA and 0.05 mM Melittin, pH 7.0,to form the dissociated/oxidized sample. The dissociated/oxidized samplewas diluted to 1-30 ng/ml (total protein concentration) in assay bufferand assayed (protocol A, Example 2) with the complementary antibodypairs shown in Table 13′. The same procedure was used to form and assaythe dissociated/reduced sample except that the dissociation and assaybuffers contained also 1.5 mM DTT to reduce the intramolecular disulfideof troponin I.

To show that the dissociation procedure did not harm free troponin Iwith respect to assays of free troponin I, purified oxidized troponin I(Bio-Tech International) was treated using the procedure to form thedissociated/oxidized sample and was then assayed with ten differentcomplementary antibody pairs (pairs 19-28 in Table 13). The assayresults (not shown) were compared with those obtained on purifiedoxidized troponin I that was not treated with the dissociationprocedure. No significant difference between the assay results for thetreated and the untreated purified troponin I was observed for any ofthe ten tested antibody pairs.

The antibody pair (#29, Table 13) consisting of biotinylated 9B1 antitroponin T monoclonal and Goat anti troponin I peptide 3 specificpolyclonal conjugate antibodies does not recognize troponin I that isdissociated from the ternary complex (Example 15), which is consistentwith the zero assay slope obtained for this antibody pair with thedissociated/oxidized and dissociated/reduced samples.

The data (Table 13) show that antibodies directed to troponin I can begenerated and selected to form an immunoassay that gives essentially thesame assay response (slope) for troponin I in the ternary complex, thedissociated/oxidized and the dissociated/reduced samples (for example,antibody pair #17). Thus, an antibody pair such as #17 could be used tomeasure the total concentration of troponin I in a sample containing allof the forms of troponin I that were tested. The procedure used togenerate and select the antibodies produces antibodies that are goodcandidates for use in an assay that measures oxidized and reduced freetroponin I and troponin I in the ternary complex in the blood ofpatients who have suffered a myocardial infarction.

TABLE 13 Assay Slope (OD₄₉₀ per ng/ml troponin I) Ternary DissociatedDissociated Pair # Conjugate Antibody Biotinylated Antibody Complex(oxidized) (reduced) 1 Goat anti troponin I Recombinant anti 0.78 0.721.23 Peptide 3 Specific troponin I #4 2 Goat anti troponin I Recombinantanti 0.77 0.55 0.53 Peptide 3 Specific troponin I #16 3 Goat antitroponin I Recombinant anti 1.07 0.77 0.77 Peptide 3 Specific troponin I#19/33 4 Goat anti troponin I Recombinant anti 0.58 0.67 1.03 Peptide 3Specific troponin I #28/36 5 Goat anti troponin I Recombinant anti 0.730.51 0.52 Peptide 3 Specific troponin I #49 6 Goat anti troponin IRecombinant anti 0.70 0.61 0.98 Peptide 3 Specific troponin I #50 7 Goatanti troponin I Recombinant anti 0.89 0.66 0.69 Peptide 3 Specifictroponin I #51 8 Goat anti troponin I Recombinant anti 0.55 0.86 0.89Peptide 3 Specific troponin I #53 9 Goat anti troponin I Recombinantanti 0.21 0.12 0.18 Peptide 3 Specific troponin I #26 10 Goat antitroponin I Recombinant anti 0.54 0.38 0.46 Peptide 3 Specific troponin I#27 11 Goat anti troponin I Recombinant anti 0.78 0.58 0.60 Peptide 3Specific troponin I #29 12 Goat anti troponin I Recombinant anti 0.600.52 0.64 Peptide 3 Specific troponin I #31 13 Goat anti troponin IRecombinant anti 0.78 0.64 0.58 Peptide 3 Specific troponin I #32 14Goat anti troponin I Recombinant anti 0.23 0.54 0.45 Peptide 3 Specifictroponin I #54 15 Goat anti troponin I Recombinant anti 0.23 0.52 0.47Peptide 3 Specific troponin I #55 16 Goat anti troponin I Recombinantanti 0.69 0.99 1.05 Peptide 3 Specific troponin I #56 17 Goat antitroponin I Recombinant anti 1.02 1.08 1.08 Peptide 3 Specific troponin I#57 18 Goat anti troponin I Recombinant anti 0.39 0.90 0.95 Peptide 3Specific troponin I #58 19 Recombinant anti Recombinant anti 0.48 0.521.06 troponin I #49 troponin I #4 20 Recombinant anti Recombinant anti0.21 0.41 0.96 troponin I #49 troponin I #28/36 21 Recombinant anti Goatanti Troponin I 1.18 1.12 1.25 troponin I #49 Peptide 3 Specific 22Recombinant anti Recombinant anti 2.42 3.31 7.63 troponin I #51 troponinI #4 23 Recombinant anti Recombinant anti 1.20 2.52 5.69 troponin I #51troponin I #28/36 24 Recombinant anti Recombinant anti 1.33 0.53 1.20troponin I #50 troponin I #16 25 Recombinant anti Recombinant anti 1.930.81 1.95 troponin I #50 troponin I #19/33 26 Recombinant anti Goat antiTroponin I 1.92 1.52 2.12 troponin I #50 Peptide 3 Specific 27Recombinant anti Recombinant anti 1.58 3.33 3.49 troponin I #53 troponinI #16 28 Recombinant anti Recombinant anti 1.60 3.75 4.28 troponin I #53troponin I #33/79 29 Goat anti Troponin I 9B1 anti troponin T 0.47 0.000.00 Peptide 3 Specific monoclonal

Example 24 Effect of Protamine and Melittin on the Non-SpecificAbsorption of Troponin to Latex Particles

Avidin-HS coated magnetic latex (Example 1) was washed and resuspendedto 1% solids in three different diluents: serum (Hybritech Inc., SanDiego, Calif.), serum containing 0.2 mg/ml protamine chloride and serumcontaining 0.1 mM melittin. A 125 μl volume of each resuspended latexsolution was mixed with a 125 ul volume of serum containing either 6ng/ml of oxidized purified troponin I (Bio-Tech International, Inc) or18 ng/ml of purified ternary troponin complex to form a solution oftroponin and latex. Also, a 125 μl volume of serum, serum containing,0.2 mg/ml protamine chloride or serum containing 0.1 mM Melittin wasmixed with a 125 ul volume of serum containing either 6 ng/ml ofoxidized purified troponin I or 18 ng/ml of purified ternary complex totorn a solution of troponin without latex. The solutions were incubatedfor 30 minutes at room temperature. The latex was pelleted and thesupernatants collected. The supernatants from the solutions of troponinand latex and the solutions of troponin without latex were assayed fortroponin (Protocol A, Example 2, except serum was used in place of assaybuffer) using antibody pair #17 (Table 13) to determine theconcentration of troponin. For each diluent, the fraction of troponinrecovered in the supernatants of the solutions of troponin and latex(Table 14) was determined by dividing the measured concentration oftroponin in the supernatant from the solution of troponin and latex bythe measured concentration of troponin in the solution of troponinwithout latex.

The data show that both melittin and protamine chloride increase therecovery of troponin, indicating that melittin and protamine chloridereduce the non-specific absorption of troponin to the latex.

TABLE 14 Fraction of Troponin Recovered in the Supernatant Troponin FormNo Addition +Protamine Chloride +Melittin Free Oxidized 0.49 0.73 0.80Troponin I Troponin Complex 0.58 0.87 0.77

Example 25 Effect of Protamine and Melittin on the Assay Response of aSandwich Immunoassay for Troponin that Utilizes Latex Particles

Avidin-HS magnetic latex (Example 1) was washed and resuspended to 1%solids in two different diluents; assay buffer and assay buffercontaining 0.1 mM melittin. In the wells of a microtiter plate, 25 μl ofeach resuspended latex solution was mixed with 25 μl of assay buffercontaining purified oxidized troponin I (Bio-Tech International) atvarious concentrations between 0 and 16 ng/ml to thou a solution oftroponin I and latex. The solutions were incubated at room temperaturefor 30 minutes. To each well was added 50 μl of a solution consisting ofthe two antibodies of pair #17 (Table 13), each at 2.5 ug/ml, to form areaction mixture. The reaction mixture was incubated for 15 minutes andthe latex was then pelleted, washed and further treated as described inProtocol A (Example 2). The results are expressed in Table 15 as anassay slope with units of OD₄₉₀ per ng/ml troponin I.

The data show that the addition of melittin to the assay increases theassay response by about a factor of two, thus increasing assaysensitivity to troponin I. In data not shown, incorporation of protamine(at 0.03-0.1 mg/ml) and melittin (at 0.017-0.05 mM) into an immunoassayfor troponin I that utilized latex particles increased the assayresponse by factors of 10% to 400%.

TABLE 15 Additive Assay Slope None 0.021 Melittin 0.040

1. A cardiac troponin assay method, comprising: measuring theconcentration of cardiac troponin I in a body fluid sample obtained froma human subject using an immunoassay for cardiac troponin I on said bodyfluid sample, wherein said immunoassay uses antibodies comprising: (a) afirst antibody that specifically binds to uncomplexed cardiac troponinI, wherein said first antibody also specifically binds to cardiactroponin I complexed to troponin T and troponin C; or (b) another firstantibody that specifically binds to cardiac troponin I, uncomplexed toeither troponin T or troponin C and a second antibody that specificallybinds to cardiac troponin I, complexed to troponin T and troponin C; or(c) a combination thereof; and wherein said immunoassay is configured toprovide assay results that are within a factor of two for (i) cardiactroponin I, uncomplexed to either troponin T or troponin C, at aconcentration between 1 and 15 ng/mL; and (ii) an equivalentconcentration of cardiac troponin I in a complex with troponin T andtroponin C.